CHARACTERIZATION OF THE BREVENAL BINDING SITE: AN ALLOSTERIC SITE AFFECTING VOLTAGE SENSITIVE SODIUM CHANNELS SITE 5
Elena P. Gold
A Dissertation Submitted to the University of North Carolina Wilmington in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
Department of Biology and Marine Biology
University of North Carolina Wilmington
2010
Approved by
Advisory Committee
Dr. Andrea J. Bourdelais Dr. Henry Jacocks
Dr. Stephen Kinsey Dr. Richard Satterlie
Dr. Robert Roer Dr. Daniel G. Baden Chair
Accepted by
Dean, Graduate School TABLE OF CONTENTS
ABSTRACT ...... v
DEDICATION ...... vi
ACKNOWLEDGMENTS ...... vii
LIST OF TABLES ...... viii
LIST OF FIGURES ...... ix
INTRODUCTION ...... 1
Voltage-sensitive (voltage-gated) Na+ Channels: Structure and Function ...... 3
Receptors Located on Voltage Sensitive Sodium Channels ...... 12
Neurotoxins Specific for Voltage-Sensitive/Voltage-Gated Na+ Channels ...... 13
Radiolabeled Probes and Properties for Receptors on VSSC ...... 17
Interactions between receptor sites ...... 20
Background of Study including Aims and Objectives ...... 23
MATERIALS AND METHODS ...... 26
Materials ...... 26
Receptor Binding Assays ...... 26
Electrophysiology: Patch Clamp Studies ...... 36
RESULTS ...... 47
Receptor Binding Assays ...... 47
Electrophysiology: Patch Clamp Studies ...... 67
DISCUSSION ...... 90
Receptor Binding Studies ...... 90
Automated Patch Clamp Studies ...... 95
iii
CONCLUSIONS ...... 101
Receptor Binding ...... 101
Electrophysiology of N2a and HEK 293 NaV1.4 cells ...... 101
REFERENCES ...... 103
iv
ABSTRACT
The aldehyde moiety of brevenal, a natural compound produced by Karenia
brevis, was reduced to the alcohols, brevenol and 3H-brevenol. Confirmation of the
structures of brevenol and 3H-brevenol was performed using high performance liquid
chromatography and nuclear magnetic resonance. The binding brevenal, brevenol and
3H-brevenol were investigated in respect to brevetoxin binding at receptor site 5 of
mammalian voltage sensitive sodium channels (VSSCs). Brevetoxins are polyether
neurotoxins produced by the marine dinoflagellate, K. brevis. Analysis of tritiated
brevetoxin-3 (3H-PbTx-3) binding to its receptor site within rat brain synaptosomes
generated a dissociation constant (KD) of ~2.6nM, while results suggested a KD of 68 nM for 3H-brevenol binding. Inhibition studies show that, PbTx-2 and 3, brevenal and
3 brevenol inhibited H-PbTx-3 binding and inhibition constants (Kis) of 1.2 nM, 2.5 nM, 97
nM, and 661 nM were obtained for each ligand respectively. However, inhibition studies
3 with H-brevenol demonstrated of Kis of 75 nM and 57 nM for brevenal and brevenol but
no inhibition of this novel probe by PbTxs 2 and 3. The data from receptor binding
studies suggested that a simple model of mutual competitive exclusion did not exist between the brevetoxins and brevenal receptor site. No other site specific ligand for
sites 1-5 and 7 on VSSCs displaced 3H-brevenol binding. Both brevenal and brevenol,
when applied at a concentration of 10 nM, exhibited similar activity to PbTx-2 in some
experiments performed on two types of VSSCs, a neuronal cell line (N2a) and in human
kidney cells transvected with muscle sodium channel isoforms (HEK 293 NaV1.4).
These results support the proposal that brevenal and its synthetic derivatives bind to an uncharacterized receptor site associated with voltage sensitive sodium channels.
v
DEDICATION
I would like to dedicate this thesis to my family and my husband whose continued support and encouragement along the way have meant more to me than they will ever know.
vi
ACKNOWLEDGEMENTS
Along this journey, I have been blessed by many individuals. I want to acknowledge my advisor, Dr. Daniel G. Baden and committee members, especially Drs.
Henry “Karl” Jacocks and Andrea Bourdelais. They spent many hours, days, months and years listening to all my suggestions, talking to me and challenging me to be a better scientist. For their undying dedication, I am forever grateful. I also want to acknowledge my parents, Pamela and Robert L. Perrineau Sr., who encouraged me continually to pursue education even before they received their own college degrees.
Finally, I acknowledge “Uncle” Thomas Riley, whose insightful words and pursuit of a higher education and a doctoral degree inspired me to complete my own.
vii
LIST OF TABLES Table Page
1. Neurotoxins that target VSSCs and their corresponding receptor sites ...... 14
2. Summary of radioprobes for receptors on VSSC, their corresponding Dissociation constants (concentration at which half the maximal binding occurs) and binding maximum (maximum receptor sites per tissue preparation) ...... 21
3. Binding Characteristics of 3H-brevenol in the presence of unlabeled brevenal or brevenol ...... 53
4. Summary of displacement of 3H-brevenol by known sodium channel ligands ...... 69
5. Summary of the effects of 10 nM brevenal, 10 nM brevenol and 10nM PbTx-2 on properties of VSSC using N2a cells and HEK 293 transfected with NaV1.4 channels ...... 89
viii
LIST OF FIGURES
Figure Page
1. Structure of the polyether brevetoxins (PbTxs) produced by the marine dinoflagellate, Karenia brevis (formerly Ptychodiscus brevis and Gymnodinium breve) ...... 2
2. Amino acid sequence similarity and phylogenetic relationships by maximum parsimony analysis of rat sodium channel sequences NaV1.1- NaV1.9 and NaX ...... 6
3. Membrane topology of the rat brain voltage-gated sodium channel ...... 7
4. Sequences of S4 units (a) an III-IV linkers (b) from sodium channels of rat brain type II, Drosophila, Electrophorus electric organ, giant axons of squids Liligo bleekeri and L. opalescens, and of the SNC4A gene from human muscle ...... 9
5. Synthesis of brevenol and 3H-brevenol ...... 27
6. Purification of brevenal reduction reaction involves an initial separation on a C18 HPLC column (Varian: Dynamax) UV absorbance was measured at 215 nm ...... 29
7. HPLC chromatogram of Group I (Figure 2) run on a reversed-phase phenyl-hexyl column using UV absorbance at 215 nm ...... 30
8. HPLC chromatogram of Group II (Figure 2) run on a reversed-phase phenyl-hexyl column ...... 31
Graph 1. Double Reciprocal plot used for enzymes and substrate complexes ...... 37
Graph 2. Mathematical Model of Lineweaver-Burk Plot also known as Double Reciprocal Plot ...... 38
9. Example of the INa-IV curve protocol used on Nanion Port-A-Patch whole cell automated patch clamp system ...... 42
10. Example of the inactivation protocol for Nanion Port-A-Patch automated patch clamp system in whole cell mode ...... 44
11. Example of recovery from inactivation protocol using Nanion Port-A-Patch automated planar clamp system (NPC-1 chips) ...... 46
ix
12. Proton NMR spectra for HPLC peaks I and II (Figures 3 and 4, Materials and Methods) ...... 48
13. Results of a typical saturation receptor binding experiment of 3H-PbTx-3 to rat brain synaptosomes. (Inset) Scatchard plot of specific binding is shown for visualization purposes ...... 49
14. (A) Specific saturation binding of 3H-brevenol to rat brain synaptosomes. (B) Scatchard plot for visualization of binding data ...... 51, 52
15. Competitive inhibition of 3H-PbTx-3 binding to site 5 (VSSC) by brevenol ...... 55
16. Noncompetitive Inhibition of 3H-PbTx-3 by brevenal ...... 56
17. Reduction of 3H-brevenol binding as a percent of the specific binding using unlabeled competitors: PbTx-2 (site 5), PbTx-3 (site 5), brevenal, and brevenol ...... 57
18. Competitive Inhibition of 3H-brevenol versus brevenol ...... 58
19. Competitive Inhibition of 3H-brevenol by brevenal ...... 59
20. Effect of tetrodotoxin (site 1) on 3H-brevenol binding ...... 60
21. Effect of veratridine (site 2) on 3H-brevenol binding ...... 62
22. Effect of aconitine (site2) on 3H-brevenol binding ...... 63
23. Effect of Leiurus quinquestriatus (site 3) venom on 3H-brevenol binding ...... 64
24. Effect of Centruroides sculpturatus (site 4) venom on 3H-brevenol binding ...... 65
25. Reduction of 3H-PbTx-3 binding as a percent of the maximum specific binding using unlabeled competitors: PbTx-2, PbTx-3, brevenal, brevenol ...... 66
26. Effect of amiloride on 3H-brevenol binding ...... 68
27. The effect of 10 nM brevenal on Na-IV relationship in N2a cells was taken using a holding potential of -120 mV. (B) Conductance-voltage (G-V) relationships determined from the peak Na+ currents for cells treated with 10 nM brevenal ...... 70
28. (A) The effect of 10 nM brevenol on Na-IV relationship in N2a cells was taken using a holding potential of -120 mV. (B) Conductance-voltage (G-V) relationships determined from the peak Na+ currents for cells treated with 10 nM brevenol ...... 71
x
29. (A) The effect of 10 nM PbTx-2 on Na-IV relationship in N2a cells was taken using a holding potential of -120 mV. (B) Conductance-voltage (G-V) relationships determined from the peak Na+ currents for cells treated with 10 nM PbTx-2 ...... 72
30. Example of how the kinetic activation and inactivation were determined from the maximum current ...... 74
31. (A) Effect of 10 nM brevenol, brevenal and PbTx-2 on the kinetic Activation of the sodium current in N2a cells. (B) Effect of 10 nM brevenal, brevenol and PbTx-2 on tau in N2a cells, represented a percent change from control ...... 75
32. (A) Effect of 10 nM brevenol, brevenal and PbTx-2 on the kinetic inactivation of the sodium current in N2a cells. B) Effect of 10 nM brevenal, brevenol and PbTx-2 on the time constant, tau, for kinetic inactivation, represented as percent change from control ...... 76
33. Effect of 10 nM brevenol, brevenal and PbTx-2 on the voltage dependent inactivation in N2a cells ...... 78
34. Effect of 10 nM brevenol, brevenal and PbTx-2 on the voltage dependent recovery from inactivation in N2a cells ...... 79
35. (A) The effect of 10 nM brevenal on Na-IV relationship in HEK 293 Nav1.4 cells was taken using a holding potential of -120 mV. (B) Conductance-voltage (G-V) relationships determined from the peak Na+ currents for cells treated with 10 nM brevenal ...... 81
36. (A) The effect of 10 nM brevenol on Na-IV relationship in HEK 293 Nav1.4 cells was taken using a holding potential of -120 mV. (B) Conductance-voltage (G-V) relationships determined from the peak Na+ currents for cells treated with 10 nM brevenol ...... 82
37. (A) The effect of 10 nM PbTx-2 on Na-IV relationship in HEK 293 Nav1.4 cells was taken using a holding potential of -120 mV. (B) Conductance-voltage (G-V) relationships determined from the peak Na+ currents for cells treated with 10 nM PbTx-2 ...... 83
38. (A) Effect of 10 nM brevenol, brevenal and PbTx-2 on the kinetic activation of the sodium current in HEK 293 Nav1.4 cells. (B) Effect of 10 nM brevenal, brevenol and PbTx-2 on the time constant, tau, for kinetic activation, represented as percent change from control ...... 84
xi
39. A) Effect of 10 nM brevenol, brevenal and PbTx-2 on the kinetic inactivation of the sodium current in HEK 293 with Nav1.4 cells. (B) Effect of 10 nM brevenal, brevenol and PbTx-2 on the time constant, tau, for kinetic inactivation, represented as percent change from control ...... 85
40. Effect of 10nM brevenol, brevenal and PbTx-2 on voltage dependent inactivation of whole cell patch clamp recordings in HEK 293 Nav1.4 cells ...... 87
41. Effect of 10 nM brevenol, brevenal and PbTx-2 on the voltage dependent recovery from inactivation in HEK 293 Nav1.4 cells ...... 88
42. Interactions between cell surface receptors, ligands and inhibitors ...... 93
43. The voltage sensitive sodium channels alternate between three states: closed, open and inactivated ...... 96
xii
INTRODUCTION
Blooms of the marine dinoflagellate, Karenia brevis, result in Florida Red tides in
the Gulf of Mexico. Red tides have been associated with massive fish kills, marine
mammal poisoning and human health effects. Karenia brevis (formerly Gymnodinium
breve and Ptychodiscus brevis) produces a family of neurotoxins called brevetoxins that
bind to site 5 associated with voltage sensitive sodium channels (VSSCs) (Poli et al.
1986). Brevetoxins (PbTxs) have two structural classes; PbTx-1 (brevetoxin A) type
and PbTx-2 (brevetoxin B) type toxins (Figure 1) (Baden and Mende 1982, Chou and
Shimizu 1982, Lin et al. 1981, Shimizu et al. 1986). A natural antagonist to brevetoxin,
called brevenal, was isolated from K. brevis cultures (Wilson strain) and natural blooms
was shown to competitively displace tritiated brevetoxin in a synaptosome receptor
binding assay (Bourdelais et al. 2004), an assay selective for site 5 of VSSC. The
physiological effects of brevetoxin and brevenal are also antagonistic. While
brevetoxins are lethal to fish, brevenal has been shown to be non-lethal and have the
ability to protect fish from the effect of brevetoxins in fish bioassays (Bourdelais et al.
2005). In asthmatic sheep, aerosolized brevenal has been shown to inhibit
bronchoconstriction of the lungs caused by inhaled brevetoxin at concentrations in the
pmol/mg tissue range (Abraham et al. 2005). This study also examined tracheal mucus
velocity, a marker of mucociliary clearance in sheep and results demonstrated that picomolar concentrations of brevenal alone improved tracheal mucus velocity (TMV)
(brevetoxins decrease TMV) to the degree seen with millimolar concentrations of the sodium channel blocker amiloride, the current treatment to increase tracheal mucus velocity in patients with cystic fibrosis (Hirsh et al. 2004). Suggesting that the effect of
1 Brevetoxin B backbone: Brevetoxin A backbone:
PbTx-2, R=CH2C(=CH2)CHO PbTx-1, R=CH2C(=CH2)CHO PbTx-3, R=CH2C(=CH2)CH2OH PbTx-7, R=CH2C(=CH2)CH2OH PbTx-5, [PbTx-2], C-37 O Ac PbTx-10, R=CH2CH(CH3)CH2OH PbTx-6, [PbTx-2], C27, 28 Epoxide
PbTx-8, R=CH2COCH2Cl PbTx-9, R=CH2CH(CH3)CH2OH
Figure 1: Structure of the polyether brevetoxins (PbTxs) produced by the marine dinoflagellate, Karenia brevis (formerly Ptychodiscus brevis and Gymnodinium breve)
(Rein et al. 1994).
2 brevenal on tracheal mucus velocity in the lung of asthmatic sheep is 100,000 times more potent than the effect of amiloride.
The antagonistic nature of brevenal to brevetoxin toxicity has also been seen in several in vitro studies. For example, brevenal inhibits PbTx-2 induced calcium (Ca+) influx and cytotoxicity in cerebellar granule cells in a concentration dependent manner
(LePage et al. 2007). Brevenal reduces the effect of PbTx-2 on cell proliferation of
CHO-K1-BH4 cells (Sayer et al. 2006). Brevenal protects against DNA damage induced by brevetoxins 2, 3 and 9 (from varying sources) in human lymphocytes (Sayer et al. 2005).
Brevenal is the first natural, nontoxic (based on reported physiological studies) ligand that displaces PbTx from receptor site 5 of VSSC. The possibility of using this molecule as a probe to elucidate the complex nature of the interaction of neuronal receptor sites or for the identification of a novel site on VSSC has made this compound a subject of great interest.
Voltage-sensitive (voltage-gated) Na+ Channels: Structure and Function
Voltage-gated (voltage-sensitive) sodium channels (VGSC, VSSC) open as the electrical potential differences across the cell membrane increase from a more negative voltage (~-70 mV) to a less negative potential. Closing of the channel transpires when a voltage change in the opposite direction occurs. The relationship between the membrane potential and ionic currents was first demonstrated when Hodgkin and
Huxley (1952) studied the nature of the nerve impulse. Aidley and Stanfield (1996) described Hodgkin and Huxley’s findings, noting that, “They could not detect this charge
3 movement at that time whereas the currents produced by ion movement were readily
observed, so they deduced there must be many ions moving across the membrane for
each movable membrane charge.” In order to obtain this mass movement of ions
across the membrane, there existed “active patches”, which were later characterized as
voltage–gated sodium and potassium channels.
In the normal resting state (when no change in the membrane potential has
occurred) of neuronal cells or cells in other excitable tissues, the concentration of Na+ ions is lower inside the membrane as compared to the outside and the reverse is true for the concentration of K+ ions. Hodgkin and Huxley (1952) demonstrated that the
differences in cell membrane potential are primarily due to an increase in the
membrane’s Na+ permeability. The difference in cell membrane potential is a result of
the selective permeability of the membrane to sodium, potassium and chloride ions. At rest, potassium ions (K+) can cross through the membrane relatively easily. Also at rest,
chloride ions (Cl-) and sodium ions (Na+) have a more difficult time crossing. The
change in membrane potential or a depolarizing event across the membrane results in
the net movement of the Na+ ions across the membrane through VSSCs, then a
delayed increase in K+ permeability, followed by hyperpolarization. Hyperpolarization is
the result of the net movement of K+ ions outside the cell coupled with the movement of
Cl- into the cell. In the open state of the channel, Na+ ions are able to pass through at a rate of 107-109 ions per second per channel under normal physiological conditions,
implying that there are only weak interactions between the ions and the channels
(Catterall 1984). The change in Na+ permeability consists of two phases, called
activation and inactivation. Activation of VSSCs involves the increase in Na+
4 permeability following membrane depolarization and inactivation involves the returning
of VSSC Na+ permeability to its resting value during the depolarized stage. It takes
longer for K+ channels to open. When they do open, K+ rushes out of the cell, reversing
the depolarization. Also at about this time, Cl- passes into the cell through chloride
channels and Na+ channels start to close. This causes the membrane potential to go
back toward its resting potential of -70 mV (a repolarization). The membrane potential actually repolarizes above -70 mV (a hyperpolarization) because the potassium channels stay longer than necessary to achieve the resting potential.
There are many types of VSSCs which control the movement of Na+ ions through
the cell membrane (Figure 2). The basic composition of VSSCs in several mammalian
species, other vertebrate species including fish and many insect species is an alpha (α)
subunit which is approximately 260 kiloDaltons (kDa), associated with auxiliary beta (β)
subunits (Catterall 2005). Specifically, the VSSC of the rat brain (the model system
used in this research) has two auxiliary β subunits, β1 and β2 (Keynes 1994). Beta
subunits modulate channel gating and regulate the level of channel expression in the
plasma membrane (Isom 2001). They have also been shown to function in cell adhesion
by having interactions with the extracellular matrix and the cytoskeleton (Isom 2001).
The amino acid sequence for VSSC was first determined by Noda and
colleagues using cDNA prepared from the electric eel organ messenger RNA (Noda et
al. 1984). They found that the protein chain is 1820 amino acid residues long and consists of four homologous domains with very similar sequences. Each α-subunit consists of these four helical domains each with six transmembrane segments (Figure
3). The four domains are assumed to be clustered together within the cell membrane
5
Figure 2: (incorporated from Catterall et al. 2005) Amino acid sequence similarity and phylogenetic relationships by maximum parsimony analysis of rat sodium channel sequences NaV1.1-NaV1.9 and
NaX. Amino acid sequences were aligned using CLUSTALW, replaced with published nucleotide sequences and subjected to analysis using PAUP. The tree constructed by showing common ancestry with invertebrate sodium channel sequences, although they are not shown in the figure.
NaV1.1 is found in central neurons, the cell bodies and cardiac myocytes. NaV1.2 is located in central neurons in premyelinated and nonmyelinated axons. NaV1.3 is distributed in early development of neurons, prenatal life, cell bodies of neurons and cardiac myocytes. NaV1.4 is found in skeletal muscle. NaV1.5 channels are located in cardiac myocytes, immature and denervated skeletal muscle and some brain neurons. NaV1.6 have a somatodendritic distribution throughout output neurons of the brain, Purkinje cells, brain stem and spinal cord, astrocytes and node of Ranvier of sensory and motor axons of the peripheral nervous system. NaV1.7 is in Schwann cells, sympathetic neurons and neuroendocrine cells. NaV1.8 is abundant in dorsal root ganglion (DRG) neurons and their axons.
NaV1.9 are preferentially expressed in DRG nociceptive (pain receptors) neurons.
6
Figure 3: Membrane topology of the rat brain voltage-gated sodium channel
(incorporated from Catterall et al. 2005). Groups of cylinders represent the four proposed domains each containing S1-S6 transmembrane segments. β1 and β2 extracellular domains are shown as immunoglobin-like folds; ψ denotes glycosylation sites; and P, indicates phosphorylation sites by protein kinase A (circles) and protein kinase C (diamonds). The h (in shaded circle) represents site of inactivation particle in the inactivation loop. S4 voltage sensors are denoted with ++. Finally, the sites of α- scorpion toxin, β-scorpion toxin, batrachotoxin (BTX) and brevetoxin (PbTx) receptor location are also denoted on this diagram.
7 and form a cylindrical pattern with an aqueous pore along its central axis. Evidence
favoring this arrangement includes a study performed using antibodies that bind to part
of the S5-S6 link in domains I and IV which interfere with the binding of scorpion
toxins, suggesting these two domains are adjacent to each other (Thomsen and
Catterall, 1989). Each fourth transmembrane segment is positively charged at every
third amino acid residue, consisting of either an arginine or a lysine (Figure 4). Between the arginine and lysine residues are mostly nonpolar residues; many organisms show homology among their sequences including the rat brain, electric eel and the human muscle sodium channel gene SNC4A (Figure 4; Keynes 1994) . The idea of the S4 segment as a voltage sensor was originally proposed by Armstrong in 1981. Five years later Catterall proposed the sliding helix model (Catterall 1986) for the S4 segment.
Catterall’s model of the sliding helix demonstrated that the S4 segment responds to depolarization by rotating ~60° and moving outward bringing a positive charge of the S4 segment in line with the negative charge of parallel segments. The net result is a negative charge inside the membrane and a positive charge outside which is equivalent to the movement of one charge across the membrane (Catterall 1985, 1996).
Confirmation of the sliding helix hypothesis was shown using site-directed mutagenesis, by Stuhmer and colleagues (1989), when they replaced the arginine and lysine residues from the S4 segments of domains I and II of rat sodium channels and injected the resultant mRNAs made from the altered cDNAs into Xenopus oocytes. These mutant channels were expressed in the oocyte membrane and were investigated by voltage clamping patches of the membrane. Results of these experiments indicated that the steepness of the relation between the channel opening and the membrane potential
8
Figure 4: (incorporated from Keynes 1994) Sequences of S4 units (a) an III-IV linkers
(b) from sodium channels of rat brain type II, Drosophila, Electrophorus electric organ,
giant axons of squids Liligo bleekeri and L. opalescens, and of the SNC4A gene from human muscle. Positively charged residues (denoted by a plus (+) sign) of lysine (K), arginine (R), and histidine (H) are in regular type but negatively charged residues
(shown with a minus (-) sign), aspartic (D) and glutamic acid (E) are ringed. All sequences show homology for VSSC regions despite the fact that sequences are from mammalians, insects and invertebrates.
9 was progressively reduced as the positively charged S4 segment residues were
replaced by neutral or negatively charged residues (Stuhmer et al. 1989).
Another important feature of VSSC/VGSC is the ability to inactivate after and
during depolarization. Inactivation is the phenomenon where channels become non-
conductive while the membrane is still depolarized. Site direct mutagenesis in the
cytoplasmic loop (Figure 1) connecting domains III and IV demonstrated a reduction in
the rate of inactivation (Aidley 1996). This region contains a cluster of positively
charged residues and 3 adjacent hydrophobic residues (I, F, M; IFM particle) which
appear to be vital to the inactivation process.
Although all VSSCs function in a similar manner, 10 different subtypes have been identified. Different types of VSSC are classified based on their differences in amino acid sequence (Catterall et al. 2005) and are predominant in specific tissue types. Nevertheless, amino acid sequences of all VSSC have a common origin, which are hypothesized to share common ancestry with insect VSSC sequences (Figure 2).
VSSC have a wide distribution in the central and peripheral nervous systems but are also located in skeletal muscle and cardiac myocytes. In general, and predictably,
VSSCs are highly concentrated in cells that function in neuronal activity, muscle activity and cardiac function.
In addition to nerve conduction and muscle contraction, many important physiological processes are dependent upon the normal activity of VSSC. Alterations in function of VSSC result in a number of known diseases. For example, three similar inherited diseases are produced by mutations in skeletal muscle Na+ channel gene
(NaV1.4 is the sodium channel type). Examples are hyperkalaemic periodic paralysis
10 (HYPP), paramyotonia congenita (PC), and potassium aggravated myotonia (PAM)
(Barchi 1995; Hoffman et al. 1995; Cannon 1996). HYPP is brought on by heavy
physical work and is associated with an increase in cellular potassium. The myotonias,
PC and PAM, occur when VSSC do not inactivate fully and patients show intermittent
muscle stiffness or involuntary contraction triggered by cold conditions (in the case of
PC) or consumption of potassium rich food, such as bananas (specific for PAM).
While the majority of the receptor sites located on VSSC are characterized by the
binding of neurotoxins, one site is a target for many local anesthetics. Two examples of
anesthetics that involve the VSSC are procaine and lidocaine which act by blocking
sodium channels at a receptor located in the fourth domain near the sixth
transmembrane segment. They are lipid soluble and reach their binding site by first
dissolving in the lipid phase of the membrane (Aidley 1996). This anesthetic site is also
the site for binding of many antidepressants, anticonvulsants and antiarrythmics.
Another group of sodium channels, epithelial sodium channels (ENaC), is the
primary target of the drug amiloride. The potency of brevenal in the lung has been
shown to be much higher than amiloride in an asthmatic sheep model (Abraham et al.
2005). Because of the association between brevenal and amiloride, ENaCs are of great interest for this thesis. ENaCs function as the rate-limiting step for sodium absorption across airway epithelia, which in turn regulates airway surface liquid (ASL) volume and the efficiency of mucociliary clearance (Donaldson et al. 2002). ENaC has been shown to be regulated by protasin, a serine protease in JME-CF15 cells, a cystic fibrosis (CF) airway epithelial cell line (Tong et al. 2004). While CF is an autosomal recessive disorder that results in mutations in the CF transmembrane conductance regulator gene
11 (CFTR: functions as an apical epithelial chloride channel), dysregulation of sodium
transport is proposed to play a major role in the pathophysiology of CF lung disease
(Donaldson and Boucher 2007). Initially, amiloride was used extensively in in vitro and
in human studies. Despite experimental evidence that amiloride improves mucociliary
clearance (Kohler et al. 1986), clinical trials of inhaled amiloride did not yield healthy enhancement of lung function (Donaldson and Boucher 2007; Knowles et al. 1990;
Graham et al. 1993). Because brevenal is known to improve mucociliary clearance, similar to amiloride, determining its molecular target is of great interest for its potential role in treating diseases like CF and asthma.
Receptors located on Voltage Sensitive Sodium Channels
Seven receptor sites for voltage sensitive sodium channels have been characterized by the toxins/compounds that bind to them (Wang and Wang 2003).
These receptors are termed orphan site because no ligand endogenous to mammalian systems has been found for these sites that have such a profound effect on sodium channel function. All of the known pharmacological agents that act on VSSC have receptor sites on the α-subunit (Catterall et al. 2005). Although these sodium channels are primarily mediated by a change in cellular membrane potential, binding of endogenous or exogenous compounds to receptors associated with VSSC/VGSC can also cause changes in the function of the channels (Baden et al. 1998; Catterall and
Gainer 1985; Catterall and Risk 1981; Catterall et al. 2005; Sharkey et al. 1987;
Thomsen and Catterall 1989). Like many other receptors, those located on the
VSSC/VGSC are membrane-localized with a two-fold function: (1) recognizing a ligand
12 with a great deal of sensitivity and chemical selectivity and (2) converting the process of
recognition into a signal that results in cellular action (Yamamura et al. 1985).
Neurotoxins Specific for Voltage-Sensitive/Voltage-Gated Na+ Channels
Because of the high degree of selectivity and avidity with which neurotoxins bind
to receptors on VSSC, they have been used as probes to determine the structural and
functional properties of these sodium channels. The physiological effects of such
neurotoxins have been confirmed by electrophysiology experiments. Site 1 toxins
include tetrodotoxin (TTX), saxitoxin (STX) and -conotoxins (Table 1) (Catterall et al.
1979; Krueger et al. 1979). These toxins are known to inhibit sodium ion conductance.
While there are several available sources for TTX, including frogs, octopus and the
goby, it was first isolated from liver and reproductive tract of puffer fish (Narahashi et al.
1960). STX is produced by a marine dinoflagellate of the genus Gonyaulax (Riegel et
al. 1949; Sommer and Meyer 1937) and µ-conotoxins are isolated from the cone snail
(Cruz et al. 1985; Mcintosh et al. 1982). Binding of TTX and STX involves interactions
between guanidine residues within these molecules and the outer pore of the α-subunit
of the sodium channels (Baden et al. 1995; Krueger et al. 1979; Noda et al. 1989;
Ritchie and Rogart 1977). Therefore, binding of these toxins results in a flat position on
the surface of the membrane, like a “lid” on the channel (Baden 1983; Baden et al.
1995). Conformation of a single site for TTX and STX was shown from studies involving
the direct competition of radiolabeled 3H-TTX versus unlabeled STX and, subsequently,
the blocking of 3H-STX by unlabeled TTX (Colquhoun et al. 1972; Barnola et al. 1973;
Henderson et al. 1973).
13 Table 1: Neurotoxins that target VSSCs and their corresponding receptor sites (adapted from Wang and Wang 2003) Receptor site Neurotoxins Physiological effects Putative Location Tetrodotoxin Inhibition of Na+ Saxitoxin p‐loop at D1, D2, 1 permeability µ‐Conotoxin D3, D4
Batrachotoxin Persistent activation, D1‐S6 Veratridine depolarization of D2‐S6 2 Aconitine resting potentials, D3‐S6 Grayanotoxin repetitive firings D4‐S6
α‐Scorpion toxins Prolonged Na+ channel D4:S3‐S4 3 sea anemone opening Loop δ‐Atracotoxins D4:s5‐S6
β‐Scorpion Shifts in activation D2: S3‐S4 4 Toxins gating; repetitive Loop firings Brevetoxins Shifts in activation D1:S6 5 Ciguatoxins gating D4:S5
Prolonged Na+ channel 6 δ‐Conotoxins opening ?
Persistent activation, D1‐S6 DDT depolarization of 7 D2‐S6 Pyrethroids resting potentials, D3‐S6 repetitive firings Local anesthetics, Inhibition of Na+ D1‐S6 8 anticonvulsants, permeability D3‐S6 antiarrythmics, D4‐S6 antidepressants
14 Site 2 specific toxins include veratridine, batrachotoxin, grayanotoxin and aconitine (Table 1) (Catterall et al. 1981; Wang and Wang 2003). Varying sources exist for these molecules. Batrachotoxin is a steroidal alkaloid from the skin of several
Columbian frog species (Daly and Witkop 1971). Veratridine is also a steroidal alkaloid that is isolated from plants of the family Liliaciae (Benforado 1967). Aconitine and grayanotoxins are produced by Aconitum napellus and flowering plants of the genus
Rhododendron (and also related species of the family Ericaceae), respectively. Site 2 specific toxins cause a persistent activation of the sodium channel. The persistent activation is the result of a combination of the blockage of inactivation of the channels and the shifting of the activation voltage threshold to more negative potentials which causes activation at normal resting potential (Cestele and Catterall 2000; Wang and
Wang 2003). As inferred by radioligand receptor binding studies in rat brain synaptosomes, batrachotoxin is a full agonist at this site while all others are only partial agonists (Brown et al. 1981; Ray et al. 1978).
Site 3 toxins include -scorpion toxins (from the venom of African scorpions, subfamily Buthinae) and sea anemone, these toxins inhibit inactivation of VSSC. The binding of scorpion toxins is dependent upon a cell membrane potential being similar to that of sodium channel during activation (approximately -60 mV) and not the normal resting membrane potential of the cell (Catterall 1977; Ray et al. 1978; Catterall 1979;
Tamkun and Catterall 1981). On the other hand, site 4 toxins, -scorpion toxins from the venom of American scorpions of the subfamily Centrurinae, shift the activation voltage threshold of VSSC to more negative values but have no effect on inactivation. Unlike site 3 toxins, binding of site 4 toxins is not dependent on a membrane potential similar
15 to one observed during activation of VSSC potential (Cestele and Catterall 2000). A
sixth site has been identified by the selective binding of -conotoxin to VSSCs.
Although no physiological effect has been associated with its binding in mammals, -
conotoxin inhibits VSSC inactivation in mollusks (Fainzilber et al. 1994, 1995). The
seventh site is characterized by the binding of pyrethroids like deltamethrin and DDT
(Lombet et al. 1988).
Finally, receptor site 5 on VSSC/VGSC is a binding site for both brevetoxins and
ciguatoxins. Ciguatoxins are polyethers isolated from several types of fish from
Caribbean regions, Hawaii, and subtropical and tropical areas worldwide including, but
not limited to, barracuda, amberjack, horse-eye jack, black jack, and other large species
of jack, king mackerel, large groupers, and snappers. However, ciguatoxins are
produced by the dinoflagellate Gambierdiscus toxicus (Bagnis et al. 1980; Satake
1993). Ciguatoxin direct radiolabeling has not been performed but its binding has been
inferred though displacement of radiolabeled brevetoxins, most notably tritiated
brevetoxin-3 (3H-PbTx-3).
Brevetoxins (PbTxs), produced by the Florida red tide dinoflagellate Karenia brevis (formerly Gymnodinium breve and Ptychodiscus brevis), bind to receptor site 5
associated with VSSC (Poli et al. 1986). Binding of brevetoxins to VSSC elicits four
effects: 1) shifting the activation voltage threshold to more negative values, 2) prolonged
channel open time, 3) inhibition of inactivation and 4) the induction of subconductance
states for Na+ across the channel (Jeglitsch et al. 1998). No studies have indicated a
competition or inhibition of PbTx binding by other toxins occupying other receptor sites affecting sodium channel function. Earlier reports of brevetoxin binding indicate that
16 PbTx-1 does not interfere with binding at sites 1-3 (Catterall and Risk 1981) and has no
interaction with site 4 (Catterall and Gainer 1985). Moreover, Sharkey and colleagues
(1986) showed that PbTx-2 has no effect on neurotoxin binding at site 1 and 3 although
allosteric modulation enhances binding at site 2 and 4. While there is modification in
binding at other sites by brevetoxin, again there is no direct competition. Therefore, this
evidence led to the conclusion that brevetoxins bind to a unique site (site 5) associated
with Na+ channels. Confirmation of PbTx binding at a novel site was demonstrated by
determining that tritium-labeled PbTx-3 (3H-PbTx-3) specific binding was unaffected by
the addition of toxins acting on sites 1-4; however, PbTx-2, inhibits the specific binding
of 3H-PbTx-3 in rat brain synaptosomes (Poli et al. 1986). A summary of the neurotoxin
receptor sites and mode of action is shown in Table 1.
Radiolabeled Probes and Properties for Receptors on VSSC
Radioligand receptor binding studies using tissue preparations yield important
information about the specificity of the ligand for its target receptor. These studies have
proved essential to determine the number and distribution of channels in excitable
tissues and to elucidate more information about the protein components of the channels
themselves. Two values obtained from these studies include the dissociation binding constant (KD) and the maximum number of specific receptor sites (Bmax) (Yamamura et
al. 1978). The KD is used to describe the affinity between a ligand (here we use
neurotoxins) and a protein (VSSC). In other words, KD is a measure of how tightly a
ligand binds to a protein in a reversible manner. The dissociation constant has molar
units (M), which correspond to the concentration of ligand at which half of the binding
17 sites on a particular matrix are occupied. The smaller the KD, the more tightly bound the ligand is, or the higher the affinity between ligand and protein. On the other hand, the
Bmax is the maximal number of binding sites which is approached asymptotically. Bmax is
the density of the receptor in the tissue being studied. Both the KD and the Bmax are
determined using nonlinear regression analysis as the radioligand concentration is increased.
Remember that site 1 specific toxins are STX, TTX and µ-conotoxin. Both STX and TTX have been labeled with tritium ([3H]) to study its properties at this receptor site.
3 H-Saxitoxin, was shown to have a KD of 1.7 nM and a Bmax= 4.9 pmol/mg of protein
(Catterall et.al. 1979). Additional experiments from Catterall and colleagues revealed a
3 KD of 2.3 nM for H-Saxitoxin and a Bmax for this ligand as 4.8 pmol/mg of protein.
Including two other studies, values for the maximum binding capacity of 3H-Saxitoxin
ranged from 1.4 pmol/mg of protein to 4.9 pmol/mg of protein (Catterall 1979; Krueger
3 et al. 1979; Jacques et al. 1978). In addition to this, the KD for H-TTX and derivatives
(TTX- glycine, lysine, and β-alanine) ranged from 3.2 to 5.1 nM. These values for 3H-
TTX are slightly higher than those reported before but comparable to a KD equal to 4.8
nM for 3H-STX demonstrated in another study (Chicheportiche et al. 1980).
Nevertheless, the differences in values for KD and Bmax between Catterall (and colleagues) and Chicheportiche and colleagues is attributed (by Catterall) to the differences in preparation, purity and specific activity of the radioactive product.
The most well characterized probe for receptor site 2 of VSSC is the tritiated derivative of batrachotoxin (3H-batrachotoxin). 3H-batrachotoxin-A 20-α-benzoate has
several KD values depending on the unlabeled ligand used in assays to determine
18 3 nonspecific binding. H-batrachotoxin-A 20-α-benzoate exhibited a KD of 0.05µM, 7µM,
and 1.2µM. in the presence of the unlabeled ligands batrachotoxin, veratridine and
aconitine, respectively (Brown et al. 1981; Catterall et al. 1981). In the presence of 1
3 µM scorpion toxin, H-batrachotoxin A 20-α-benzoate exhibits a KD = 82 nM with a
binding capacity of 2.1 pmol/ mg of protein (Catterall et al. 1981). The higher KD value obtained in the presence of scorpion toxin can be attributed to the fact that scorpion toxin binds at another receptor site on VSSCs and allosterically modifies the batrachotoxin site.
125 For receptor site 3, scorpion mono [ I] iodotoxin, was determined to have a KD
of 2.2 nM in rat brain synaptosomes and a Bmax= 0.594 pmol/mg of protein (Ray et al.
1978). Additionally, experiments from Catterall and colleagues (1979) indicated the binding of 125I- scorpion (monoiodo) toxin (prepared from Leiurus quinquestriatus
venom) to a single class of binding sites with a KD of 1.9 nM and a binding maximum of
1.3 pmol/mg of protein. Total estimates of scorpion toxin binding capacity range from
0.3 pmol/mg of protein to 1.7 pmol/mg of protein (Morrow and Catterall 1978; Vincent et
al. 1980; Ray et al. 1978; Jover et al. 1978, 1980; Catterall et al. 1981). Comparatively,
125I-AaH II (scorpion alpha toxin II), binding in rat brain synaptosomes demonstrated a
KD= 0.3 nM and Bmax= 0.32 pmol/mg of protein (Cestele et al. 1995).
Site 4 and 5 radioligands exhibit similar KD but have significantly different binding
maximums for rat brain synaptosomal preparations. Receptor binding studies for site 4
using 125I-labelled Centruroides suffusus suffusus (Css II) indicated a single class of
binding sites with high affinity (KD= 3-5 nM) and a Bmax= 0.8-1.4 pmol/mg of protein in
rat brain synaptosomes (Jover et al. 1980). Moreover, 3H-PbTx-3, the ligand for site 5,
19 was found to have a binding affinity of 1-7 nM (Poli et al. 1986) and a higher number of
binding sites than most other known receptors. In rat protein preparations, the Bmax values ranged from 6 to 13.5 pmol/mg of protein (Poli et al. 1986). Finally, the radioactive probe for receptor site 6 is a δ-conotoxin labeled by radio-iodination (125I-
δTxVIA). This ligand was found to have a KD of 1.9 nM and a Bmax of 0.67 pmol/mg of
protein (Fanzilber et al. 1994). A summary of the KD and Bmax values for receptor sites
1-6 are shown in Table 2 for comparison to values obtained during this research project.
Interactions between receptor sites
Allostery is a term referring to the modification of one binding site as the result of
binding to a different binding site by its ligand. Allosteric regulation is often associated
in biochemistry with the regulation of enzymes but the basic principles are applied in
binding of ligands to receptors. Those ligands which, when bound to their respective
active sites, enhance the binding of another ligand to its active site are called allosteric
activators, whereas those which decrease the activity are called allosteric inhibitors.
Several ligands act as allosteric modulators on other receptor sites when bound. For
example, site 1 neurotoxin saxitoxin, enhances the binding of α-scorpion toxin for its
receptor site (Ray et.al. 1978). Likewise, batrachotoxin, veratridine and aconitine are
allosteric activators of scorpion toxin binding on site 3 (Catterall 1979 and Ray et al.
1978). Veratridine positively modulates the binding of 125I-Aah II to site 3 (Cestele et al.
1995). This binding is prevented in the presence of PbTx-1.
Binding of site 3 toxins (sea anemone toxin and α-scorpion toxin) enhances
sodium channel activation caused by site 2 toxins (list) (Catterall and Beress 1978). In
addition to this, specifically, ATX II (sea anemone toxin)and ScTx II (another site 3
20 Radioactive Receptor Site Dissociation Binding Constant (K ) Maximum (B ) Reference Probe on VSSC D max
Catterall 1979; Chicheportiche et al. 3 1.4-4.9 pmol/mg of H-Saxitoxin Site 1 1.7-4.8 nM 1980 ; Krueger et al. protein 1979; Jacques et al. 1978 3 2.1 ± 0.2 pmol/mg H- Site 2 80-700 nM Catterall et al. 1981 Batrachotoxin of protein Morrow and Catterall 125 1.4-1.7 pmol/mg of [ I] ScTx Site 3 3-8 nM 1978; Catterall et al. i protein 1979
3 6-13.5 pmol/mg of Site 5 1-7 nM Poli et al. 1986 H-Brevetoxin protein 125 0.67 ± 0.06 [ I] δTxVIA Site 6 1.9 ± 0.3 nM Fainzilber et al. 1994 i pmol/mg of protein
Table 2: Summary of radioprobes for receptors on VSSC, their corresponding dissociation constants (concentration at which half the maximal binding occurs) and binding maximum (maximum receptor sites per tissue preparation). The sources for the information are provided in the reference column.
21 ligand) cause a decrease in the amount of veratridine needed for half maximal
activation of sodium channels as determined using sodium flux studies. When ATXII
and ScTX II is bound at saturation concentrations of veratridine, there is an increase in
activation of VSSCs resulting in a shift of veratridine binding at site 2 toward being a full
agonist for its receptor under these conditions much like batrachotoxin. Therefore, since
binding of site 2 neurotoxins affects binding of site 3 neurotoxins and vice versa, it is
concluded that the interactions between neurotoxin receptor site 2 and 3 is bidirectional
and allosteric.
PbTx-1 is an allosteric inhibitor of site 3 since it reduces the specific binding of
125I-Aah II, the most potent known α-scorpion toxin, in rat brain sodium channels
(Cestele et al. 1995; Baden 1989). Binding of TTX at site 1 reverses the allosteric
inhibition of PbTx on site 3 of VSSCs (Cestele et al. 1996). However, PbTx-2 had no
effect on Leiurus quinquestriatus quinquestriatus V, another type of α-scorpion toxin, or on saxitoxin binding to their VSSC receptors in rat brain synaptosomes (Sharkey et al.
1987). These findings suggest that receptor site 5 interacts with site 3 depending on
the site 3 specific ligand used, but this receptor site does not interact with site 1.
Binding at site 1, however, can modify the effect that site 5 ligand binding has on site 3.
On the other hand, PbTx-1 and PbTx-2 augment the binding of batrachotoxin and
veratridine at receptor site 2 of VSSC (Catterall and Risk 1980; Sharkey et al. 1987;
Trainer et al. 1993). Finally, PbTx-2 increases the binding of Centruroides suffusus
suffusus (CsTx II) to site 4 of VSSC (Sharkey et al. 1987). Therefore, allosteric
interactions that exist among receptor sites (5 and 2, and also 4 and 5) are very complex.
22
Background of Study including Aims and Objectives
A novel compound, brevenal, produced by Karenia brevis has been shown to
compete with receptor sites occupied by 3H-PbTx-3 at concentrations over a hundred-
fold greater than the concentration of brevetoxin used to inhibit its binding (Bourdelais et
al. 2004). The displacement of brevetoxin by brevenal suggests two possibilities; 1)
brevetoxin and brevenal share the same receptor site on site 5 of voltage sensitive
sodium channels or 2) they do not share the same binding site but brevenal binding to
the brevenal receptor site modulates the brevetoxin binding site in a way to reduce
binding of brevetoxin.
The physiological effects of brevetoxin and brevenal are generally antagonistic to
each other. While brevetoxins are lethal to fish, brevenal has been shown to be non-
lethal and have the ability to protect fish from the effect of brevetoxins in fish bioassays
(Bourdelais et al. 2005). In asthmatic sheep, aerosolized brevenal (at pmol/mg tissue
concentrations) has been shown to inhibit bronchoconstriction of the lungs caused by
subsequently inhaled brevetoxin (Abraham et al. 2005). Abraham and colleagues
(2005) also examined tracheal mucus velocity, a marker of mucociliary clearance, in
sheep demonstrated that picomolar concentrations of brevenal alone improved tracheal
mucus velocity (TMV) (brevetoxins decrease TMV) to the degree seen with millimolar concentrations of the sodium channel blocker amiloride, a compound used currently to
improve mucus transport in people with cystic fibrosis, suggesting that brevenal may be
100,000 times more potent than amiloride in the lung.
23 Brevenal is a natural ligand, with no reported toxic effects, that displaced 3H-
PbTx binding in rat brain synaptosomes (Bourdelais et al. 2004). The possibility of using this molecule as a probe to elucidate the complex nature of the interaction of neuronal receptor sites or for the identification of a novel site has made this compound a subject of great interest. Identification of receptors by reversible binding of labeled agonists and antagonists to intact cell and cell membrane preparations is a conventional method for receptor identification for biochemical purposes (Yamamura et al.1985).
Other methods used to obtain information about VSSC include photoaffinity labeling
using autoradiography for molecular weight information about a receptor (Poli
dissertation 1985); using molecular genetics which allows for the examination of the
protein at the amino acid level (Catterall et al. 2005); or site directed mutagenesis
(Stuhmer et al.1989). No one technique is “better” or is going to yield a complete solution to the problem of understanding the function of the sodium channel in terms of its molecular structure (Levinson et al. 1986). Each method, however, provides a clue to the nature of sodium channel function and structure. Therefore, brevenal binding characteristics were investigated and a receptor binding assay for brevenal was developed.
The work presented here has several objectives: first, to design a radioligand receptor assay for the binding of brevenal to rat brain synaptosomes by using a partial synthetic derivative, 3H-brevenol; second to characterize the nature of 3H-brevenol
binding with respect to saturability, binding affinity, and maximum number of receptor
sites per tissue preparation; third, to confirm binding of brevenal and brevenol
(brevenal/ol) is associated with VSSCs and determine how brevenal/ol affects VSSCs
24 using electrophysiology experiments; and finally, compare 3H-Brevenol binding to other known VSSC specific neurotoxins and to characterize the site in relation to those known receptor sites 1-7 of VSSCs.
25 MATERIALS AND METHODS
Materials
Brevenal, PbTx-1, PbTx-2 and PbTx-3 were purified from unialgal cultures of
Karenia brevis (Wilson strain) as previously described (Bourdelais et al. 2004). Tritiated
3 sodium borohydride (NaB H4 50-80 Ci/mmole) was purchased from Amersham
Pharmacia (St. Louis, MO) or American Radiolabeled Chemicals (St. Louis, MO). The
methods for reduction of PbTx-2 to PbTx-3 and 3H-PbTx-3 and purification methods
were described previously (Poli 1985). 3H-PbTx-3 was supplied from the Baden
laboratory. All unlabeled competitors for displacement experiments were purchased from Sigma–Aldrich Chem. Co. (St. Louis, MO). All other compounds were reagent grade or better and were obtained from commercial sources. Frozen whole rat brains
(male; Sprague-Dawley) were purchased from Harlan (Indianapolis, IN). External, seal enhancer, and internal buffer recipes and cell preparation methods for electrophysiology studies were provided by Nan]i[on (München, Germany). Electrophysiology data was
analyzed by an analysis of variance (ANOVA) using SAS (SAS 9.1.3 from SAS Institute,
Inc.).
Receptor Binding Assays
Preparation and Characterization of Brevenol and Tritium-labeled Brevenol (3H- Brevenol)
Brevenol was prepared by reduction of the aldehyde on brevenal to the
corresponding alcohol using NaBH4 (Figure 5). All solvents were dried by storage over
molecular sieve (4 Angstrom) before use. NaBH4 was dissolved in dimethylformamide
26 H3C
O H HO
H CH 3 O O H H OH
H
O
H Brevenol CH 3
O
H3C H to the aldehyde H to the aldehyde 3
H3C
CH 3
4
]
H
[ OH
3
B
3
l
a . The reaction reduces the aldehyde
C
N
e
r
o
C
4
H
B
a
N
OH 2 H-brevenol 3 brevenal with the addition of a
H3C
O HO H
H CH 3 O
O s of brevenol and H H OH H
O
H CH 3
O Brevenal
H3C
H3C Figure 5: Synthesi functional group in brevenal to an alcohol. Synthesis of tritiated brevenol results the functional group in brevenal to an alcohol. Synthesis reduction of the aldehyde group on carbon.
CH 3
O
27 (DMF) to a final concentration of 0.4 M and added to a reaction vessel containing 3-5
mg of brevenal and a thirty-fold molar excess cerium chloride. The reaction was
performed at room temperature with constant stirring for 1 hour in methanol. The reaction was stopped by the addition of 1 mL acetone. The product mixture was washed using diethyl ether and distilled water (50:50, v:v), retaining the product in the ether phase. The synthesis of 3H-brevenol paralleled the synthesis of unlabeled
3 brevenol, by substituting NaB H4 for NaBH4.
The purification/identification of brevenol was performed as follows. The diethyl
ether layer was dried using a rotary evaporator and the dried product was dissolved in
acetone to be purified using high performance liquid chromatography (HPLC). The
acetone product mixture was injected onto a reverse phase column (Varian; Dynamax
microsorb 100-5 C18 column 10 mm x 250 mm, (Palo Alto, CA); the mobile phase was
MeOH:H2O, 90:10, v:v, and the flow rate was 3.4 mL/min) and the elution of products
were monitored using ultraviolet absorbance at 215 nm. The two resultant groups, labeled Group I and II were collected separately as they eluted from the column (Figure
6). The two fractions were then re-purified by reversed-phase HPLC (Phenomenex;
Luna phenyl-hexyl column, 5 m, 100A, 4.6 mm x 250 mm; MeOH:H2O; 90:10, v:v, 1.6
mL/min) and monitored at a UV absorbance of 215 nm. Group I from the initial C18 column (Figure 2) yielded two peaks (mobile phase 90% MeOH:10% water, v:v). Peak I eluted at 4 min and Peak II eluted in 5 min (Figure 7). Group II (Figure 6), from the C18 column, yielded three peaks on the phenyl hexyl column, peak I (4 min), peak II at 5 min and peak III at 6 min (Figure 8). Both peaks eluting at 4 min (Figure 7 and 8) from the
phenyl hexyl column were combined and dissolved in benzene (C6D6) for NMR
28
Group 1
Group 2
Figure 6: Purification of brevenal reduction reaction involves an initial separation on a
C18 HPLC column (Varian: Dynamax) UV absorbance was measured at 215 nm.
Injection of the reaction mixture produced multiple peaks in 2 groups. Each group was collected in separate vials and subsequently run on a reversed-phase phenyl hexyl column.
29
Peak I (4 min)
Peak II (5 min)
Figure 7: HPLC chromatogram of Group I (Figure 2) run on a reversed-phase phenyl- hexyl column using UV absorbance at 215 nm. The two resultant peaks I and II had retention times of 4 min and 5 min, respectively.
30 Peak II (5 min)
Peak I (4 min)
Peak III (6 min)
Figure 8: HPLC chromatogram of Group II (Figure 2) run on a reversed-phase phenyl- hexyl column. The resultant three peaks had retention times of 4 min, 5 min and 6 min.
Peak I and II were analyzed by NMR using a 5.0 mm TXI probe in C6D6 and were found to be brevenol and brevenal respectively.
31 spectroscopy. Peaks that eluted at 5 min denoted on Figure 3 and 4 were also combined and dissolved in (C6D6) for NMR spectroscopy (Bruker Avance 500 MHz
system using either a 5.0 mm TXI or a 5.0 mm BBO probe) experiments: 1H, 13C, 1H-13C
HMBC, 1H-13C HSQC, 1H-1H COSY and 1H-1H ROESY to identify the fraction
containing brevenol.
The purification of the reduction reaction product of brevenal to 3H-brevenol performed by Dr. Jacocks paralleled the protocol for the unlabeled reduction previously described. However, 3H-brevenol purification and identification was conducted based
on the retention times of brevenol (unlabeled) reduction products as seen on similar
C18 and phenyl hexyl HPLC columns within the tritium facility. The NMR was
unavailable for use in identification of the 3H-brevenol. To further verify the identity of
the 3H-brevenol, brevenol (non-radioactive) was mixed with 3H-brevenol and run on the
HPLC C18 column (mobile phase: 90:10, MeOH:H2O) at a UV absorbance of 215 nm;
identity being adjudged as co-elution of the two compounds.
Preparation of Synaptosomes
The preparation of synaptosomes was modified from Dodd et al. (1981). Ten
frozen male rat brains were thawed on ice and subsequently homogenized in 10 mL of
ice-cold homogenization buffer (0.32 M sucrose, 0.005 M sodium phosphate, and a
protease inhibitor cocktail of 1 mM iodoacetamide, 0.1 mM phenylmethylsulfonyl fluoride
(PMSF), 1 mM (1,10) phenanthroline and 1 M pepstatin A, brought to pH 7.4 with
H3PO4) with ten strokes of a motor driven Teflon/glass homogenizer. The resulting
homogenate was sedimented at 700 x g for 10 min. The supernatant was saved and
32 the pellet was resuspended in 10 mL homogenization buffer and homogenized again.
Sedimentation was repeated at 700 x g as above. The second supernatant was combined with the first and the pellet was discarded. The supernatant mixture was layered over 3 mL of 1.2 M sucrose solution (containing the above protease inhibitor cocktail) in 10 mL centrifuge tubes and centrifuged at 105,000 x g for 30 min at 4 C.
The material at the interface, between the 0.32 M and 1.2 M sucrose solutions, was
collected, minimizing the amount of 1.2 M sucrose solution included. The material was
layered over 0.8 M sucrose solution containing protease inhibitors and centrifuged at
140,000 x g for 35 min at 4 C in 5 mL centrifugation tubes. The final pellet containing
synaptosomes was resuspended to a concentration of approximately 1 mg protein/mL in standard binding medium (SBM) (50 mM HEPES, 130 mM choline chloride, 5.4 mM
KCl, 0.8 mM magnesium sulfate, 5.5 mM glucose, 1 mM EGTA and the protease inhibitor cocktail and brought up to pH 7.4 with Tris base) and frozen at -80 °C for use in subsequent assays. Protein concentration of the synaptosomal preparations was measured spectrophotometrically using the Bradford protein assay (1976) purchased from BioRad (Hercules, CA) which is based on an absorbance shift in the dye
Coomassie with (0-1.8 mg/mL) BSA as the standard.
Saturation Binding of 3H-PbTx-3 and 3H-brevenol
Saturation binding of 3H-PbTx-3 or 3H-brevenol was measured independent of
each other using a rapid centrifugation technique as described by Poli (1985). All
buffers and equipment in contact with the brains/homogenates were at ice temperature
except during centrifugation. All experiments were performed in the SBM with 1 mg/mL
33 bovine serum albumin (BSA) (SBM + BSA). No detergent was used because studies
indicated that too much nonspecific binding (high background) occurred when 3H- brevenol was combined in solution with detergent. Synaptosomes (0.05 mL) were added to each reaction tube with a mixture containing 0.05 mL of 3H-PbTx-3 (~10 to 0.1
nM) or 3H-brevenol (~300 to 2 nM) and 5 µL of (10 nM or 50 nM) unlabeled effectors
(e.g. PbTx-2, PbTx-3, brevenol or, brevenal, as appropriate) in 0.395 mL SBM + BSA.
All unlabeled effectors were incubated with the synaptosomal preparation for 30 min.
Subsequently, 3H-brevenol was added. After vortexing and incubation at 4 C for 0.5
hour, all samples were centrifuged for 2 minutes at 14,000 x g then returned to the ice
bath. The supernatants in all tubes were individually aspirated and rapidly washed with
2-3 drops of wash medium (163 mM choline chloride, 5 mM HEPES, 1.8 mM calcium
chloride, 0.8 mM magnesium sulfate, 1 mg/mL BSA, and brought up to a pH of 7.4
using Tris base). The pellets were transferred to scintillation vials, suspended overnight
in 2.5 mL Scintiverse biodegradable scintillation fluid (Fisher Scientific) and the bound
radioactivity was determined by liquid scintillation spectrometry using the LS 6500
Liquid Scintillation Counter (Beckman Coulter; Brea, CA). Nonspecific binding was
measured in the presence of 10 M PbTx-3 for experiments performed with 3H-PbTx-3 and 50 M brevenal or 50 M brevenol for experiments using 3H-brevenol. Specific
binding was calculated as the difference between total binding and nonspecific binding.
Equilibrium dissociation constants (KD) and binding site maxima (Bmax) were determined by non-linear regression analysis by GraphPad Prism version 4.00 for Windows,
GraphPad Software, San Diego California USA, www.graphpad.com. KD and Bmax
34 values that were obtained when using brevenol or brevenal as the unlabeled ligand were compared using an unpaired Student’s t-test (two-tailed) in GraphPad Prism 4.0.
Inhibition Experiments with 3H-PbTx-3 and 3H-brevenol Probes
The displacement of each radiolabeled ligand was determined in the presence of
various competitors (ranging in concentration from10-12 to 10-5 M or 5 x 10-6 to 50
mg/ml). These experiments, like the saturation binding experiments, were done at ice
temperature using the rapid centrifugation technique above with an initial volume of 0.05
mL synaptosome suspension, 0.05 mL radioprobe (~3 nM for 3H-PbTx-3 and ~9-10 nM for 3H-brevenol), 5 L unlabeled competitor, and 395 L SBM + BSA. Displacement
experiments were performed at a fixed concentration of radioligand. Equilibrium
inhibition constants (Ki) were determined by non-linear regression analysis by
GraphPad Prism version 4.00 for Windows, GraphPad Software, San Diego California
USA, www.graphpad.com.
Double-Reciprocal Experiments
Double reciprocal experiments were carried out at 3-4 nM 3H-PbTx-3 and 9-10
nM 3H-brevenol. For each concentration of radioligand, increasing concentrations of
unlabeled ligand were employed as the potential displacing ligand. For example, at
each final concentration of 3H-PbTx-3, each triplicate contained at least two of the
following concentrations of unlabeled PbTx-2: zero (control, ethanol only), 10 µM, 25
µM or 50 µM. Concentrations were empirically defined. The reaction mixture contained a total volume of 500 µL comprised of 50 µL synaptosome suspension, 50 µL
35 radioprobe stock, and 5 L unlabeled competitor and 395 L SBM + BSA. The synaptosomal suspension and unlabeled competitor were incubated for 30 min, followed by addition of the radioprobe stock, vortexing and incubation for an additional
30 min. Afterwards, the pellets were harvested via rapid centrifugation as above. “Free” ligand concentrations were determined by pipetting a 10 µL aliquot of the supernatant.
Immediately following the aspiration of the remaining supernatant, the pellet was harvested. Then 2.5 mL of Scintiverse was added and radioactivity was determined by liquid scintillation spectrometry. The pellet DPMs resulting from rapid centrifugation yielded the “bound” radioligand values. Regression lines and intercepts were determined by linear regression analysis by GraphPad Prism version 4.00 for Windows,
GraphPad Software, San Diego California USA, www.graphpad.com. Resultant
competition data were classified as either competitive or noncompetitive using double
reciprocal plots in a classic Lineweaver-Burk treatment (1934) (Graph 1 and 2).
Electrophysiology: Patch Clamp Studies
Cell Preparation
Two cell lines, neuroblastoma N2a (ATCC CCL-131) from mice Mus musculus
and human embryonic kidney cells, HEK 293 stably expressing human muscle sodium channels (Nav1.4) (donated by Dr. Al George, Vanderbuilt University) were grown under
standard cell culture conditions in Dulbecco’s minimum essential medium (DMEM),
supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and
either the antibiotic geneticin (N2a) or G418 (HEK 293 cells). Cells were passed two days prior to experiments, and were harvested at 60% to 80% confluency. To harvest,
36
Graph 1: Double Reciprocal plot used for enzymes and substrate complexes. This
same analysis is applied to receptor-ligand binding. Graph (incorporated from
Lineweaver and Burk 1934) demonstrates competitive inhibition between line 44, 45 and 42. Non-competitive inhibition is shown as comparison between lines 43 and 42.
37 Graph 2 (A, B, C):
(incorporated from Berg et al.
2002) Mathematical Model of
Lineweaver-Burk Plot also
known as Double Reciprocal
Plot. This model was
originally used for binding of
substrates to enzymes.
However, since 1934 it has
been applied to the binding of
ligands to receptors to aid in
the determination of the nature
of binding between the two
entities.
38 cells were washed twice with magnesium- and calcium-free phosphate-buffered saline
(PBS) before being dissociated from the surface of the culture dish by the addition of 2 mL of 1x trypsin-EDTA (Invitrogen). The trypsin induced enzyme reaction was arrested by the addition of 9 mL of growth medium. The cells were spun at 100 x g for 2 min and the pellet resuspended in 2 mL of external medium (containing 140 mM NaCl, 4 mM
KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM D-glucose monohydrate, and 10 mM
HEPES/NaOH, pH 7.4). The cells were centrifuged for a second time at 100 x g for 1 min to remove any remaining growth media and resuspended in external medium to a final cell density of approximately 1.5-2 x 106 cells per mL for use in patch clamp experiments.
Patch Clamp experimental protocol
Whole cell patch clamp experiments were performed using the Nanion
(München, Germany) Port-A-Patch (NPC-1 chips) automated planar patch clamp system (Farre et al. 2007). Recordings were made at room temperature in whole cell mode. The intracellular recording solution contained: 50 mM CsCl, 10 mM NaCl, 60 mM CsF, 20 mM EGTA, and 10 mM HEPES/CsOH, pH 7.2. Whole cell patch clamp recordings were achieved using an EPC-10 amplifier (HEKA Electronik, Darmstadt,
Germany) controlled by PatchMaster software (HEKA) connected to the headstage of the Port-A-Patch. The currents were filtered using an analog 3-pole Bessel 10 kHz filter
(filter 2 = 2.9 kHz) for high frequency noise and sampled at 20-25 kHz (10 µs-50 µs sample interval depending on protocol). Whole cell recordings were obtained by pipetting 20 µL of cell suspension into the NPC-1 chamber while monitoring seal
39 resistance. Since the system is automated, after pipetting the cell suspension onto the
microchip, the Patch Control system indicated initial contact with the cell surface until an
intact cell was obtained. Pressure (suction) was applied to each cell to induce sealing
at a rate of 20 mB/sec. The patch (suction) protocol for obtaining a whole cell cycled
through several steps: wait for contact, wait for cell, improve seal 1, improve seal 2, wait for whole cell and maintain whole cell (WCR). The automated system was programmed to go to the next step if the resistance of the membrane increased 10 MΩ from the prior
step. Once the seal resistance of the membrane reached between 20-50 MΩ, three
individual 10 µL aliquots of sealing enhancer buffer (80 mM NaCl, 3 mM KCl, 10 mM
MgCl2, 2 mM CaCl2, 5 mM D-glucose monohydrate, 10 mM Hepes/NaOH, pH 7.4) were
added onto and aspirated off of the port-a-patch chip. The correlation between the
suction and the sealing behavior of the cells in each step of the protocol was watched
closely. Once a whole cell was obtained, the acquisition protocol was halted. The cell
was immediately washed (by addition onto the chip) with three 20 μL aliquots of extracellular buffer. Experiments were initiated once seals of ≥ 1 gigaOhm (GΩ) were
obtained. Seal resistances varied among batches of cells and during experiments;
however data were analyzed only from cells in which the seal remained > 400 megaOhm (MΩ). Series resistance was typically <10 MΩ and actively compensated to
65-75 % using the HEKA amplifier. Linear leak subtraction was performed offline using
the seal resistance and voltage to determine the leak current. Liquid junction potential
(LJP) across the external bath and internal pipette solutions was calculated to be 10.9
mV (PClamp, Axon Instruments). Command voltage was adjusted for LJP by
subtracting this value from the voltage step values. LJP adjusted command voltage
40 was additionally adjusted for the voltage error associated with series resistance by subtracting the voltage error from the LJP adjusted command voltage values. These adjusted command voltages were used to calculate the reversal potential of each cell although reversal potential was unaffected by LJP because there was no current being applied. Conductance-Voltage (g-V) curves were calculated from the above adjusted voltage values using the equation INa/ Epot-Erev. The resulting curves were analyzed using a Boltzmann fit to obtain half-activation voltages (Molleman 2003; Walz 2007).
Automated analysis was performed using the specialist FitMaster Analysis Software
(HEKA Elektronik).
Chemical compounds at a concentration of 10 nM (PbTx-2, brevenal and brevenol) were dissolved in DMSO (final concentration of 0.01%) and external medium.
The control contained 0.01% DMSO in external buffer. All drugs/compounds were applied by pipetting 20 µL of each on and off the cell four times prior to Na-IV protocol run to ensure cells were exposed to the correct final concentration of the compound.
Immediately following the fourth addition of each drug, the Na-IV protocol (defined below) was run.
Na-IV curves
The Na-IV protocol proceeded as follows. After obtaining whole cell recordings
(WCR), cells were voltage clamped at -120 mV before applying 20ms 10 mV voltage steps from -120 to +80 mV. Inter-sweep intervals were set at 1 sec to allow for complete deactivation (Figure 9). Resulting sodium-current voltage (INa-V) data was plotted by measuring the maximum current versus each voltage step and analyzed with Fitmaster
41
Figure 9: Example of the INa-IV curve protocol used on Nanion Port-A-Patch whole cell automated patch clamp system. Cells were voltage clamped at -120 mV and subjected to 20 ms +10 mV voltage steps for 20 sweeps. The inter-sweep interval was 1 sec.
42 software (HEKA) to obtain IV linear curve fits. Activation curves were produced by
calculating conductance-voltage (g-V) curves and analyzed with a Boltzmann fit to
obtain the half-activation (V1/2) potential of each cell (Walz 2007). The voltage at which the peak sodium current (INa) occurred was further analyzed to obtain kinetic inactivation
and activation time constants. This was performed using a single exponential fit on both the activation and inactivation portions of the current trace.
Voltage dependent inactivation protocol
To further understand how the different compounds affected Na+ channel
function, a voltage-dependant inactivation protocol was performed. This protocol was
+ used to measure potential shifts in the V1/2 in the different N2a and HEK 293 Nav1.4 Na
channels in response to 10 nM PbTx-2, brevenal and brevenol. Cells were initially held
at -120 mV and pre-pulses of +∆ 5mV were applied before a 10 ms test pulse was
applied that equaled the membrane potential at which the maximum current (I) was
obtained during the previous Na-IV protocol. After the test pulse, the cell was returned
to the holding potential for a total of 20 ms and there was a 1 sec sweep delay before
the next sweep occurred. The resulting peak INa data obtained was plotted against pre-
pulse voltage and analyzed using a Boltzmann fit yielding a V1/2 representing the
membrane potential at which half the Na+ channels in the cell were non-conducting
(Walz 2007) (Figure 10).
43
Figure 10: Example of the inactivation protocol for Nanion Port-A-Patch automated patch clamp system in whole cell mode. Cells were voltage clamped at -120 mV and subjected to a pre-pulse for 250 ms ∆+5 mV steps for a total 20 sweeps. The inter- sweep interval was 1 sec. Following each pre-pulse step, a 10 ms test pulse was applied and the cell was returned to the clamp voltage.
44 Recovery from inactivation protocol
The effect of the drugs on recovery from inactivation was investigated in the Na+
channels isoforms expressed in N2a and HEK 293 cells lines. Cells were initially held
at a command voltage of -120 mV before being subjected to a 10 ms test pulse that
equaled the membrane potential at which the maximum INa was obtained during the previous INa-IV protocol. A second test pulse was applied with an interval that increased
in 1.0 ms increments for a total of 20 sweeps. An inter-sweep interval of 1.5 sec ensured full recovery of INa (Hille 1992) (Figure 11). The INa data was plotted versus
time and was analyzed using a single exponential curve fit (FitMaster). An exponential
fit curve with a steady state value (i =1) and a maximum of 3 components is represented
(-x/Tau ) by the equation: y(x) = Amp0 + Σ Ampi e i , where the Amp are the amplitudes and
are linear and Tau are time constants which are nonlinear. The resulting fit parameters
produced time constants and slopes that provided an estimate of the rate of recovery from inactivation.
45
Figure 11: Example of recovery from inactivation protocol using Nanion Port-A-Patch automated planar clamp system (NPC-1 chips). Cells were voltage clamped at -120 mV followed by a 10 ms test pulse with 1 ms intervals for 20 sweeps.
46 RESULTS
Receptor Binding Assays
Identification and Characterization of Brevenol
Brevenol was characterized using NMR spectroscopy. Final 1H-spectra for
native brevenal, and the resultant peaks from a reduction reaction of brevenal were
compared (Figure 12). The proton spectrum of peak II from HPLC chromatograms
(Figure 7 & 8, from Materials and Methods) shows an aldehyde proton (red circles) at
10.18 ppm. Comparison of A (one peak from our reduction reaction) with C (a sample of brevenal isolated from K. brevis cultures) indicates that they are identical. Additional
unsaturated protons with chemical shifts between 5.7 and 6.2 ppm (blue circles) are
also identical in spectra A and C. However, proton spectrum of peak I (Figure 7 & 8,
from Materials and Methods) shows no aldehyde group present and differences in the
unsaturated proton region (5-6 ppm) (green circles). Thus, peak I was identified as
brevenol, a synthetic derivative of brevenal in which the aldehyde was reduced to the
corresponding primary alcohol.
Saturation Binding of 3H-PbTx-3 and 3H-brevenol Receptor Binding
Saturation binding experiments, using 3H-PbTx-3 and 3H-brevenol as reporting
ligands, were performed to determine the equilibrium dissociation (KD) and binding site
maximum (Bmax) for the tissue preparation. These experiments confirm a basic
property of receptors, that of saturable high-affinity specific binding. Saturation binding
3 experiments for H-PbTx-3 yield an apparent KD of 2.6 ± 0.3 nM (n = 3) and Bmax of 7.1+
3 0.2 pmol/mg protein (n = 3) (Figure 13) and for H-brevenol the apparent KD was 68 ± 7
47 Peak at 5 Minutes A
10 9 8 7 6 5 4 3 2 ppm
Peak at 4 Minutes B
10 9 8 7 6 5 4 3 2 ppm
Brevenal Standard C
10 9 8 7 6 5 4 3 2 ppm
Figure 12: Proton NMR spectra for HPLC peaks I and II (Figures 3 and 4, Materials and Methods). A. Proton spectrum of peak II (Figure 3 & 4, Materials and Methods) shows an aldehyde proton (red circle) at 10.18 ppm. Comparison of A with C (a sample of brevenal isolated from K. brevis cultures) indicates that they are identical. Additional unsaturated protons with chemical shifts at 5-6.2 ppm (blue circles) are also identical in spectra A and C). B. Proton spectrum of peak I (Figure 3 & 4, Materials and Methods) shows no aldehyde group present and differences in the unsaturated proton region (5-
6.2 ppm) (green circles) (personal communication with Dr. Andrea Bourdelais).
48 Inset: 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 4000 1.4 1.2
Bound/Free 1.0 0.8 3500 0.6 0.4 0.2 0.0 3000 0 1 2 3 4 5 6 7 8 Bound (pmol/mg protein)
2500
2000
1500
1000 Bound RadioactivityBound (CPM)
500
0 0 1 2 3 4 5 6 7 8 9
[3H-PbTx-3] (nM)
Figure 13: Results of a typical saturation receptor binding experiment of 3H-PbTx-
3 to rat brain synaptosomes. Binding was measured using a rapid centrifugation technique at 4°C, as described in the text. Total (■) and nonspecific (♦, 10 nM PbTx-2)
binding were measured by liquid scintillation spectrometry. Specific (□) binding is
determined by subtraction of nonspecific binding at each concentration. Each data point represents the mean + s.e.m. of triplicate determinations for each concentration.
(Inset) Scatchard plot of specific binding is shown for visualization purposes
(Scatchard 1949).
49 nM with a Bmax of approximately 7.2 ± 0.9 pmol/mg protein (n = 11) (Figure 14). There
was no significant difference between KD and Bmax values obtained from saturation
binding experiments when either brevenal or brevenol was used as the unlabeled
competitor in saturation studies (Table 3). These results suggest that the reduction of
the aldehyde function to the primary alcohol (with the concomitant addition of a non-
exchangeable tritium) produces a labeled ligand that has value in the characterization of
the brevenal binding site.
Receptor binding data analyzed by nonlinear regression was transformed for
simple visualization using Scatchard analysis1. The resultant Scatchard plot is linear.
Scatchard plots of 3H-PbTx-3 (Figure 13 inset) (r2 =0.9981) and 3H-Brevenol (Figure
14B) (r2 =0.9604) are shown for visualization purposes (Scatchard 1949).
Double Reciprocal Experiments with 3H-PbTx-3, PbTx-2, PbTx-3, Brevenal and
Brevenol
Prior studies have shown that inhibition of 3H-PbTx-3 binding is competitive for
PbTx-3 and PbTx-2 (Poli et al. 1986) and also brevenal (Bourdelais et al. 2004).
Furthermore, this reduction in of 3H-PbTx-3 binding by brevenal and brevenol were
classified as either competitive or noncompetitive using double reciprocal plots in a
classic Lineweaver-Burk treatment (1934). Double reciprocal plots graph 1/bound
(radioligand concentration) versus 1/free (radioligand concentration). Chemicals that
are competitive with the labeled ligand for its receptor will converge at the y-axis when graphed. Likewise, noncompetitive antagonists will converge at the x-axis and cross
1 Scatchard analysis converts the experimental data by graphing bound radioligand/free radioligand versus the bound radioligand on the x-axis. As a result, a Scatchard plot reveals the Bmax as the x- 1 intercept, the y-intercept is the ratio Bmax/KD and the slope is defined as -1/KD.
50 30000 A
27000
24000
21000
18000
15000
12000
9000
Bound RadioactivityBound (CPM) 6000
3000
0 0 25 50 75 100 125 150 175 200 225 250 275 300 325 [3H-Brevenol] (nM)
0.12 B
0.11
0.10
0.09
0.08
0.07
0.06
0.05 Bound/Free 0.04
0.03
0.02
0.01
0.00 0 1 2 3 4 5 6 7 8 9 Bound (pmol/mg protein)
51
Figure 14: (A) Specific3 saturation binding of 3H-brevenol to rat brain
synaptosomes. Each data point represents the mean + s.e.m. of triplicate
determinations for each concentration. Analysis of nonlinear regression of specific
3 binding yields an approximate KD of 67.5 nM and a Bmax of 7.2 ± 0.9 pmoles of H-
brevenol per mg of synaptosomal protein. (B) Scatchard plot for visualization of binding data (Scatchard 1949).
3 Total and nonspecific binding were measured by liquid scintillation techniques, the difference between the two represent specific (□) binding.
52 Table 3: Binding Characteristics of 3H-brevenol in the presence of unlabeled brevenal or brevenol.
Unlabeled K (mean ± s.e.m.) B (mean ± s.e.m.) N D max Competitor (nM) (pmol/mg protein)
Brevenal 6 69 ± 1.2 7.0 ± 1.3
Brevenol 5 66 ± 5.7 7.2 ± 0.9
There was no significant difference between the KD or Bmax values for both unlabeled
competitors determined using Student’s two-tailed unpaired t-test (p ≥ 0.05).
53 the y-axis at different concentrations (analogous to Lineweaver and Burk plots for enzyme inhibition, 1934). Our results indicate a competitive inhibition 3H-PbTx-3 by
brevenol (Figure 15) but show a noncompetitive inhibition by brevenal (Figure 16) using
a double reciprocal plot of 3H-PbTx-3 binding in the presence of increasing
concentrations of each unlabeled ligand. The nonspecific binding observed in these experiments was 20% of the total binding.
Competition Experiments with 3H-brevenol by Brevenal, Brevenol and Brevetoxin
Competition experiments with 3H-brevenol showed a unique pattern of binding.
While brevenal (Ki = 75 ± 3 nM) and unlabeled brevenol (Ki = 57 ± 2nM) (n = 3)
displaced 3H-brevenol with similar potencies, both PbTx-3 and PbTx-2 were unable to
effectively inhibit 3H-brevenol specific binding in experiments using rat brain synaptosomes (Figure 17). The inhibition of 3H-brevenol binding was competitive with
brevenol (Figure 18) and brevenal (Figure 19).
3H-Brevenol Binding is not Inhibited by Ligands Selective for Other Receptor Sites on VSSC.
No inhibition of 3H-brevenol binding was exhibited in competition experiments
involving ligands selective for receptor sites (1-5) on VSSC. Competition experiments with ligands for sites 1-5 on VSSCs were performed using rat brain synaptosomes
incubated with 9 - 10 nM of 3H-brevenol and increasing concentrations of each site-
specific ligand. Inhibition of 3H-brevenol binding by brevenol is used as a standard on
all graphs (Figures 20-24 & 26). Lack of 3H-brevenol binding inhibition by tetrodotoxin
(Figure 20) suggests that 3H-brevenol does not bind to site 1 on VSSCs. For site 2
54
Figure 15: Competitive inhibition of 3H-PbTx-3 binding to site 5 (VSSC) by
brevenol. Synaptosomes were incubated for 1 hour at 4°C with increasing
concentrations of 3H-PbTx-3 in the presence of (■) 0 nM, (□) 10 nM, (▲) 25 nM, or (∆)
50 nM brevenol. Each point represents the mean ± s.e.m. of values obtained from two independent experiments with triplicate determinations per experiment.
55
Figure 16: Noncompetitive Inhibition of 3H-PbTx-3 by brevenal. Synaptosomes were
incubated for 1 hour at 4°C with increasing concentrations of 3H-PbTx-3 in the presence
of (■) 0 nM, (□) 10 nM, or (▲) 25 nM brevenal. Each point represents the mean ±
s.e.m. of values obtained from two independent experiments with triplicate
determinations per experiment.
56 110
100
90
80
70
60
50
40 % Specific Binding 30
20
10
0 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 [unlabeled ligand], log M
Figure 17: Reduction of 3H-brevenol binding as a percent of the specific binding
using unlabeled competitors: (∆) PbTx-2 (site 5), (▲) PbTx-3 (site 5), (■) brevenal,
and (□) brevenol. Each data point represents the mean ± s.e.m. of values obtained
from three independent experiments with triplicate determinations in each experiment.
Nonspecific binding was calculated as 20% of the total binding.
57
Figure 18: Competitive Inhibition of 3H-brevenol versus brevenol. Synaptosomes
were incubated for 1 hour at 4°C with increasing concentrations of 3H-brevenol in the
presence of (■) 0 nM, (□) 10 nM, or (▲) 25 nM brevenol. Each data point represents
the mean ± s.e.m. of values obtained from three independent experiments with triplicate
determinations in each experiment.
58
Figure 19: Competitive Inhibition of 3H-brevenol by brevenal. Synaptosomes were
incubated with increasing concentrations of 3H-brevenol in the presence of (■) 0 nM, (□)
10 nM, or (▲) 25 nM brevenal. Each data point represents the mean ± s.e.m. of values
obtained from three independent experiments with triplicate determinations in each
experiment.
59 120
110
100
90
80
70
60
50
40 % Specific Binding Specific %
30
20
10
0 -15 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 [unlabeled ligand], log M
Figure 20: Effect of tetrodotoxin (site 1) on 3H-brevenol binding. Synaptosomes were incubated with ~9-10 nM 3H-brevenol and increasing concentrations of tetrodotoxin (□) or brevenol (■). Each data point represents the mean + s.e.m. of three independent experiments with triplicate determinations in each experiment. Nonspecific binding was determined to be 20% of the total binding.
60 toxins, veratridine (Figure 21) and aconitine (Figure 22) did not inhibit 3H-brevenol
binding suggesting that 3H-brevenol does not bind to receptor site 2. Furthermore, α- scorpion toxins (Figure 23) and β-scorpion toxin (Figure 24) did not inhibit the binding of
3H-brevenol, an indication that 3H-brevenol does not bind to site 3 or site 4 of VSSC,
respectively. Figures 24 and 25 show venom concentrations in mg/ml because
scorpion venom is a heterogeneous mixture of substances thus preventing
determination of molarity.
The specificity of 3H-PbTx-3 binding at site 5 associated with voltage-gated
sodium channel alpha-subunits has been established through a series of receptor
binding experiments. Inhibition of 3H-PbTx-3 binding by unlabeled PbTx-3, PbTx-2,
brevenal and brevenol (Figure 25) indicated that these ligands inhibited the radioligand
binding in a concentration-dependent manner. The Ki was determined for each
unlabeled competitor listed above2. The brevetoxins, PbTx-2 and PbTx-3 were found to
3 have a similar affinity for the H-PbTx-3 binding site, although the Ki for PbTx-2 (0.81 ±
0.4 nM) was slightly less than the Ki for PbTx-3 (1.6 ± 0.2 nM). The Ki value obtained
for PbTx-3 falls within the 1.4 nM to 3.0 nM reported by previously (Baden et al. 1998;
Purkerson-Parker et al. 2000). These results indicated that PbTx-2 was more potent at displacing 3H-PbTx-3 from its binding site than PbTx-3. The natural antagonist brevenal
(Ki = 97 ± 4 nM) has a higher affinity than the alcohol derivative, brevenol (Ki = 660 ± 2 nM) at the brevetoxin binding site (Figure 25). Although equally efficacious, brevenal and brevenol are less potent inhibitors of 3H-PbTx-3 binding than either PbTx-2 or
PbTx-3.
2 The inhibition constant (Ki) gives an indication of the affinity of a ligand to a specific site and is independent of receptor concentration. It represents the concentration at which the competitor exerts its effect on the radioligand binding.
61 125
100
75
50 % Specific Binding
25
0 -15 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 [unlabeled ligand], log M
Figure 21: Effect of veratridine (site 2) on 3H-brevenol binding. Synaptosomes were incubated with ~9-10 nM 3H-brevenol and increasing concentrations of veratridine (▲) or brevenol (■). Each data point represents the mean ± s.e.m. of values obtained from three independent experiments with triplicate determinations in each experiment.
Nonspecific binding was determined to be 20% of the total binding.
62 110
100
90
80
70
60
50
40 % Specific Binding 30
20
10
0 -15 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 [unlabeled ligand], log M
Figure 22: Effect of aconitine (site2) on 3H-brevenol binding. Synaptosomes were incubated with ~9-10 nM 3H-brevenol and increasing concentrations of aconitine (∆) or brevenol (■). Each data point represents the mean ± s.e.m. of values obtained from three independent experiments with triplicate determinations in each experiment.
Nonspecific binding was determined to be 20% of the total binding.
63 110
100
90
80
70
60
50
40
% Specific% Binding 30
20
10
0 -5 -4 -3 -2 -1 0 1 2 [unlabeled ligand], log (mg/ml)
Figure 23: Effect of Leiurus quinquestriatus (site 3) venom on 3H-brevenol binding. Synaptosomes were ~9-10 nM 3H-brevenol and increasing concentrations of
L. quinquestriatus venom (○) or brevenol (■). Each data point represents the mean ±
s.e.m. of values obtained from three independent experiments with triplicate
determinations in each experiment. Nonspecific binding was determined to be 20% of
the total binding. Notice x-axis unit is in log mg/ml.
64 110
100
90
80
70
60
50
40 % Specific Binding Specific % 30
20
10
0 -5 -4 -3 -2 -1 0 1 2 [unlabeled ligand], log (mg/ml)
Figure 24: Effect of Centruroides sculpturatus (site 4) venom on 3H-brevenol binding. Synaptosomes were with ~9-10 nM 3H-brevenol and increasing concentrations of C. sculpturatus venom (●) or brevenol (■).Each data point represents the mean ± s.e.m. of values obtained from three independent experiments with triplicate determinations in each experiment. Nonspecific binding was determined to be 20% of the total binding. Notice x-axis unit is in log mg/ml.
65 110
100
90
80
70
60
50
40 % SpecificBinding 30
20
10
0 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 [unlabeled ligand], log M
Figure 25: Reduction of 3H-PbTx-3 binding as a percent of the maximum specific
binding using unlabeled competitors: (∆) PbTx-2, (▲) PbTx-3, (■) brevenal, and (□)
brevenol. Each data point represents the mean ± s.e.m. of values obtained from three
independent experiments with triplicate determinations in each experiment. Nonspecific
binding was 20% of the total binding.
66 In addition to these findings, no inhibition of 3H-brevenol binding was shown by
amiloride (Figure 26), an epithelial Na channel (ENaC) blocker, implying that 3H-
brevenol most likely does not directly affect the amiloride binding site. A summary of
the results for the site specific ligands that bind to receptor sites on VSSC are listed in
Table 4.
Electrophysiology: Patch Clamp Studies
Automated Patch Clamp Electrophysiology: N2a (Neuroblastoma) cells Na-IV Curve Results
Sodium current-voltage (Na-IV) curve data for N2a cells was plotted by
measuring the maximum current at each voltage step via analysis by Fitmaster (HEKA)
to obtain IV linear curve fits. Na-IV curves (n=4) including the vehicle control for each
treatment, brevenal (Figure 27A), brevenol (Figure 28A) and PbTx-2 (Figure 29A) are
shown. The average current voltage for each treatment was normalized for differences in cell size by dividing the average current (pA) by the average cell size (pF).
Normalizing for cell size allows for better comparison of sodium currents in N2a cells because cells can be quite variable in size and the larger the cell the more Na channels it will have in the membrane therefore leading to a larger current for that cell. Na-IV curves were analyzed for changes in maximum current and shift in the V1/2 activation.
The activation curves were produced by calculating the conductance of the cell for each voltage step. Conductance was plotted versus voltage to obtain G-V curves. Provided that the current through a single channel is linear, conductance is proportional to the number of open channels. Thus G-V curves resemble activation curves (Walz 2007).
The G-V curves were fit using the Boltzmann equation and the V1/2 potential was
67 110
100
90
80
70
60
50
40 % Specific% Binding 30
20
10
0 -15 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 [unlabeled ligand], log M
Figure 26: Effect of amiloride on 3H-brevenol binding. Synaptosomes were
3 incubated with ~9-10 nM H-brevenol and increasing concentrations of amiloride (*) or brevenol (■). Each data point represents the mean ± s.e.m. of values obtained from three independent experiments with triplicate determinations in each experiment.
Nonspecific binding was determined to be 20% of the total binding.
68 Table 4: Summary of displacement of 3H-brevenol by known sodium channel ligands.
Displacement of 3H- Receptor Site on Compounds Brevenol VSSC/VGSC
Tetrodotoxin No displacement Site 1
Veratridine No displacement Site 2
Aconitine No displacement Site 2
Leiurus quinquestriatus No displacement Site 3 venom
Centruroides No displacement Site 4 sculpturatus venom
Brevetoxins 2 & 3 No displacement Site 5
No displacement Site 7 Deltamethrin (personal communication Dr. Henry Jacocks)
Amiloride No displacement Blocks ENaC sodium channels
Brevenal Ki =74.8 nM (new)
Brevenol Ki =56.5 nM (new)
presumably the same as brevenal
69 I‐V relationship N2a cells 60 40 20
Voltage (mV) 0 ‐150 ‐100 ‐50‐20 0 50 100 Vehicle ‐40 Brevenal ‐60
‐80
‐100 (pA/pF) Na ‐120 I
1.2 B
) 1 max 0.8 (g/g
0.6 Vehicle 0.4 Brevenal
Conductance 0.2
0 ‐111 ‐101 ‐91 ‐81 ‐71 ‐61 ‐51 ‐41 ‐31 ‐21 ‐11 ‐1 Voltage (mV)
Figure 27: (A) The effect of 10 nM brevenal on Na-IV relationship in N2a cells was taken using a holding potential of -120 mV. (B) Conductance-voltage (G-V) relationships determined from the peak Na+ currents for cells treated with 10 nM brevenal. There was a significant difference between the conductance curve shift in - - the half-activation potential for 10 nM brevenal (V1/2 = 34.5) and vehicle control (V1/2 = 24.8) treated cells (Z test = -4.802; p-value < 0.008).
70
A I‐V relationship N2a cells 60
40
20
0 Voltage (mV) ‐150 ‐100 ‐50‐20 0 50 100 Vehicle ‐40 Brevenol ‐60
‐80
‐100 (pA/pF) Na ‐120 I
1.2 B 1 )
max 0.8 (g/g
0.6 Vehicle 0.4 Brevenol Conductance 0.2
0 ‐111 ‐101 ‐91 ‐81 ‐71 ‐61 ‐51 ‐41 ‐31 ‐21 ‐11 ‐19 Voltage (mV)
Figure 28: (A) The effect of 10 nM brevenol on Na-IV relationship in N2a cells was taken using a holding potential of -120 mV. (B) Conductance-voltage (G-V) relationships determined from the peak Na+ currents for cells treated with 10 nM brevenol. There was a no statistically significant difference between the for the half- - - activation potential for 10 nM brevenol (V1/2 = 37.2) and vehicle control (V1/2 = 33.5) treated cells (Z test = -2.45; p-value > 0.008).
71 I‐V relationship N2a cells A 60
40
20
Voltage (mV) 0 ‐150 ‐100 ‐50‐20 0 50 100 Vehicle ‐40 PbTx‐2 ‐60
‐80
‐100 (pA/pF) Na ‐120 I 1.2 B 1 )
max 0.8 (g/g
0.6 Vehicle 0.4 PbTx‐2 Conductance 0.2
0 ‐111 ‐101 ‐91 ‐81 ‐71 ‐61 ‐51 ‐41 ‐31 ‐21 ‐11 ‐1919 Voltage (mV)
Figure 29: (A) The effect of 10 nM PbTx-2 on Na-IV relationship in N2a cells was taken using a holding potential of -120 mV. The visible change in maximum current is not significant (Z test = -1.31; p-value > 0.008). There is no change in the reversal potential. (B) Conductance-voltage (G-V) relationships determined from the peak Na+ currents for cells treated with 10 nM PbTx-2. There was a significant difference - between the half-activation potential for 10 nM PbTx-2 (V1/2 = 29.3) and vehicle control - - (V1/2 = 23.9) treated cells (Z test = 2.84; p-value < 0.008).
72 obtained. Conductance was calculated as peak I/(Vpot-Vrev) where Vpot is the membrane potential and Vrev is the reversal potential. Results indicated that there were no
significant differences between the maximum current of all treatments and their
respective vehicle control. The half activation (V1/2 ) potential occurred at a significantly
more negative potential for the 10 nM brevenal and 10 nM Pbtx-2 treatments compared to the vehicle control (Figures 27B & 29B), an indication that channels activate at more negative voltages when treated with these two compounds.
The time constants, tau (τ), for the kinetic activation and inactivation of cells were determined from the voltage sweep that exhibited the maximum sodium current (INa).
The kinetic activation of the cell was determined by fitting the downward slope of the current trace from the activation potential to the peak current using a 1- exponential fit algorithm (Figure 30). The kinetic inactivation of the cell was determined by fitting the upward slope of the current trace from the peak current to the reversal potential using the same 1- exponential algorithm (Figure 30). Both brevenal treated cells and PbTx-2 treated cells show a significant increase in τ (in seconds) for kinetic activation from the control (Figure 31A).The increase in the kinetic activation time constant was 27% and
21% respectively over the vehicle control (Figure 31B). On the other hand, brevenol treated cells demonstrated a significant decrease in τ (Figure 31A), a -38% change
(Figure 31B). Regarding inactivation time constants, only cells treated with PbTx-2 demonstrate a significant increase in the inactivation time constant (Figure 32A) which equated to a 15 % change over the vehicle control (Figure 32B).
73
Figure 30: Example of how the kinetic activation and inactivation were determined from the maximum current. Each part of the trace was fit to a 1-exponential algorithm to determine time constants (tau (Τ)).
74 3.00E‐04 A Kinetic Activation 2.50E‐04 * seconds) 2.00E‐04 * in
τ * ( 1.50E‐04 10 nM 1.00E‐04 Vehicle Constant 5.00E‐05
Time 0.00E+00 Brevenol Brevenal PbTx‐2
Percent change in kinetic activation from control 40.00% N2a cells * τ 20.00% * in 0.00% change
Brevenol Brevenal PbTx‐2 ‐20.00%
Percent ‐40.00%
‐60.00% * *Indicates significant difference.
Figure 31: (A) Effect of 10 nM brevenol, brevenal and PbTx-2 on the kinetic activation of the sodium current in N2a cells. Time constants (tau) obtained for all experimental treatments are significantly different from each paired vehicle control. The average time constant for brevenol treated cells was 153 µs, a value smaller than the control (247 µs) (Z test = -5.01; p-value < 0.008). The average time constant for brevenal treated cells was 179 µs, a value larger than the control = 141 µs (Z test = 5.30; p-value < 0.008). The value for tau for PbTx-2 treated cells were 198 µs on average, while the control for these cells time constant was 164 µs (Z test = 3.14; p- value < 0.008). Error bars represent + s.e.m. (B) Effect of 10 nM brevenal, brevenol and PbTx-2 on tau in N2a cells, represented a percent change from control. All treatments are significantly different from control.
75 Kinetic Inactivation A 8.00E‐04 7.00E‐04 ) τ (
6.00E‐04 5.00E‐04 * 4.00E‐04 10 nM Constant 3.00E‐04 Vehicle 2.00E‐04 Time 1.00E‐04 0.00E+00 Brevenol Brevenal PbTx‐2
Percent change in kinetic inactivation from control N2a cells 20.00% 15.00% *
τ 10.00% in 5.00% 0.00% change
‐5.00% Brevenol Brevenal PbTx‐2 ‐10.00% Percent ‐15.00% ‐20.00%
*Indicates significant difference.
Figure 32: (A) Effect of 10 nM brevenol, brevenal and PbTx-2 on the kinetic inactivation of the sodium current in N2a cells. Each cell is used as its own control. The PbTx-2 treated cells showed a significant change in inactivation time constant versus its paired vehicle control (Z test = 2.64; p-value < 0.008). The value for tau for PbTx-2 treated cells were 457 µs on average, while the control for these cells time constant was 397 µs. Error bars represent + s.e.m. (B) Effect of 10 nM brevenal, brevenol and PbTx-2 on the time constant, tau, for kinetic inactivation, represented as percent change from control. The tau for cells treated with PbTx-2 was 15% significantly different than its control.
76 Automated Patch Clamp Electrophysiology: N2a (Neuroblastoma) cells Voltage- Dependent Inactivation and Recovery from Inactivation Results
A comparison of the voltage-dependent inactivation was performed from whole cell patch clamp recordings using N2a cells. The protocol for these experiments involved holding each cell at a potential of -120 mV, prepulsing for 250 ms with increasing 5 mV steps followed by a 10 ms test pulse of -10 mV. V1/2 values, or the
voltage at which half the channels were inactivated, were determined for the following
treatments: 10 nM brevenal, 10 nM brevenol and 10 nM PbTx-2 (Figure 33). All
treatments caused a significant shift in the half inactivation potential of the channels in
the cell membrane (Figure 33). The half-inactivation potential occurred at more negative
voltage potentials compared to the vehicle control (Figure 33).
The time for recovery from voltage-dependent inactivation was determined for
cells treated with 10 nM brevenal, brevenol or PbTx-2. Experiments clamped whole
cells at -120 mV and applied a -10 mV test pulse at 1 ms intervals for 20 sweeps. The
data obtained was fit using a 1-exponential curve function to obtain the time constants
for the estimation of the rate of recovery. Cells treated with 10 nM PbTx-2 exhibited a
21% (significant) increase in the time for the rate of recovery as compared to control
(Figure 34). Brevenal and brevenol treated cells demonstrated an increase in time for recovery versus control but the changes were not statistically significant (Figure 34).
These results suggest that channels treated with PbTx-2 take a longer time to recover from inactivation but brevenal and brevenol have no affect on VSSC recovery from inactivation.
77 Voltage Dependent Inactivation N2a
Brevenol Brevenal PbTx‐2
0
‐20
‐40
(mV) ‐60 10 nM
1/2 Vehicle V ‐80
‐100 * * *
‐120 *Indicates significant difference.
Figure 33: Effect of 10 nM brevenol, brevenal and PbTx-2 on the voltage dependent inactivation in N2a cells. The average V1/2 value for cells (n=4) treated with brevenol was -89.5 mV versus -84.9 mV for control (Z test = -15.26; p-value <
0.008). The average V1/2 value for cells treated with 10 nM brevenal (n=4) was -86.2 mV - versus 80.9 mV for control (Z test = 10.96; p-value < 0.008). The average V1/2 value for PbTx-2 (n=4) is -83.6 mV and -80.2 mV for control (Z test = -11.46; p-value < 0.008). The results obtained indicate that inactivation occurs at more negative potentials for channels treated with 10 nM concentrations of brevenol, brevenal and PbTx-2. Error bars represent + s.e.m. .
78 Recovery from Inactivation N2a cells
1.00E‐02 9.00E‐03 8.00E‐03 7.00E‐03 6.00E‐03 * (s) 5.00E‐03 10 nM
Time 4.00E‐03 Vehicle 3.00E‐03 2.00E‐03 1.00E‐03 0.00E+00 Brevenol Brevenal PbTx‐2
*Indicates significant difference.
Figure 34: Effect of 10 nM brevenol, brevenal and PbTx-2 on the voltage dependent recovery from inactivation in N2a cells. Results indicate no significant change in the rate of recovery between 10 nM brevenol and brevenal and control treated cells. The time constant for recovery of 10 nM PbTx-2 was 5.84 ms versus 4.8 ms for control treated cells (Z test = 3.75; p-value < 0.008). Error bars represent ± s.e.m.
79 Automated Patch Clamp Electrophysiology: HEK 293 (Nav1.4) cells- Na-IV Curve Results
Na-IV curve data was plotted by measuring the maximum current at each voltage step via analysis by Fitmaster (HEKA) to obtain IV linear curve fits. Na-IV curves (n=4), for brevenal (Figure 35A), brevenol (Figure 36A) and PbTx-2 (Figure 37A), are shown and include the vehicle control for each treatment. The average current voltage for each treatment was normalized for differences in cell size by dividing the average current (pA) by the average cell size (pF). The activation curves were produced by calculating the conductance of the cell for each voltage step. Conductance was plotted versus voltage to obtain G-V curves. Results indicated that there was a significant decrease in maximum current from control for 10 nM brevenol and 10 nM PbTx-2 treatments. For conductance curves, the half activation potential (V1/2) occurred at a more negative potential for all treatments (10 nM brevenal, brevenol and PbTx-2) compared to the vehicle controls (Figures 35B, 36B, 37B) suggesting that sodium channels begin to activate at more negative voltages when treated with all compounds.
The time constants (τ) for the kinetic activation and inactivation from control were determined in the same manner as those obtained from N2a cells (Figure 30). Only the difference between tau (τ) of kinetic activation for brevenol treated cells was significantly different from the vehicle control (a 32% change in τ) (Figure 38A & B). For the kinetic inactivation time constants, brevenol and PbTx-2 treatments show an increase in the time constant while treatment with 10 nM brevenal demonstrates a decrease in τ (Figure
39A). Only the increase in τ for kinetic inactivation of cells treated with 10 nM PbTx-2 was significantly different from the paired controls (Figure 39B). This is an indication that cells inactivate at a more negative potential when treated with PbTx-2 but not brevenal or brevenol.
80
I‐V relationship HEK 293 Nav1.4 400
200
Voltage (mV) 0 ‐150 ‐100 ‐50 0 50100 ‐200 Vehicle ‐400 Brevenal ‐600 (pA/pF)
‐800 Na I
‐1000
1.2 B
1 )
max 0.8 (g/g
0.6 Vehicle 0.4 Brevenal Conductance 0.2
0 ‐111 ‐101 ‐91 ‐81 ‐71 ‐61 ‐51 ‐41 ‐31 ‐21 ‐11 Voltage (mV)
Figure 35: (A) The effect of 10 nM brevenal on Na-IV relationship in HEK 293
Nav1.4 cells was taken using a holding potential of -120 mV. (B) Conductance- voltage (G-V) relationships determined from the peak Na+ currents for cells treated with 10 nM brevenal. There was a significant difference between the - conductance curve shift in the half-activation potential for 10 nM brevenal (V1/2 = 59.3) - - and vehicle control (V1/2 = 54.8) (Z test = 7.54: p value < 0.008).
81 I‐V relationship HEK 293 Nav1.4
400 200
Voltage (mV) 0 -150 -100 -50-200 0 50 100 Vehicle -400 Brevenol -600 (pA/pF)
-800 Na I -1000
1.2 B
1 )
max 0.8 (g/g
0.6 Vehicle 0.4 Brevenol Conductance 0.2
0 ‐111 ‐101 ‐91 ‐81 ‐71 ‐61 ‐51 ‐41 ‐31 ‐21 Voltage (mV)
Figure 36: (A) The effect of 10 nM brevenol on Na-IV relationship in HEK 293
Nav1.4 cells was taken using a holding potential of -120 mV. A significant decrease in the maximum current is seen and a change in the reversal potential (Z test = -3.95: p value < 0.008). (B) Conductance-voltage (G-V) relationships determined from the peak Na+ currents for cells treated with 10 nM brevenol. There was a significant difference between the conductance curve shift in the half-activation potential for 10 nM - - - brevenol (V1/2 = 46.5) and vehicle control (V1/2 = 33.5) (Z test = 9.09: p value < 0.008).
82 I‐V relationship HEK 293 Nav1.4 400
200
Voltage (mV) 0 ‐150 ‐100 ‐50 0 50 100 ‐200 Vehicle ‐400 PbTx‐2 ‐600 (pA/pF)
‐800 Na I
‐1000
1.2 B 1 ) max 0.8 (g/g
0.6 Vehicle 0.4 PbTx‐2 Conductance 0.2
0 ‐111 ‐101 ‐91 ‐81 ‐71 ‐61 ‐51 ‐41 ‐31 ‐21 ‐11 ‐1 Voltage (mV)
Figure 37: (A) The effect of 10 nM PbTx-2 on Na-IV relationship in HEK 293 Nav1.4 cells was taken using a holding potential of -120 mV. A significant decrease in the maximum current is seen (Z test = -7.67: p value < 0.008). (B) Conductance-voltage (G-V) relationships determined from the peak Na+ currents for cells treated with 10 nM PbTx-2. There is a significant difference between the conductance curve shift in - - the half-activation potential for 10 nM PbTx-2 (V1/2 = 51.1) and vehicle control (V1/2 = 42.9) (Z test = -5.59: p value < 0.008).
83 4.50E‐04 A Kinetic Activation 4.00E‐04
) 3.50E‐04 τ ( 3.00E‐04 * 2.50E‐04 10 nM 2.00E‐04 constant
Vehicle 1.50E‐04
Time 1.00E‐04 5.00E‐05 0.00E+00 Brevenol Brevenal PbTx‐2 Percent change in kinetic activation from control
40% B HEK 293 Nav1.4 cells * τ 20% in
0% change
Brevenol Brevenal PbTx‐2 ‐20% Percent
‐40% *Indicates significant difference
Figure 38: (A) Effect of 10 nM brevenol, brevenal and PbTx-2 on the kinetic
activation of the sodium current in HEK 293 Nav1.4 cells. Time constant (tau) obtained only for the brevenol experimental treatment is significantly different from the vehicle control (Z test = 2.70: p value < 0.008). The average time constant for brevenol treated cells was 270 µs, a value larger than the control (204 µs) (longer time or less rapid). The average time constant for brevenal treated cells was 267 µs, a value larger than the control (344 µs), but not significant (Z test = -1.1: p value > 0.008). The value for tau for PbTx-2 treated cells (182 µs) was not significantly different from the control, whose cells time constant was 170 µs (Z test = -1.00: p value > 0.008). Error bars represent + s.e.m. (B) Effect of 10 nM brevenal, brevenol and PbTx-2 on the time constant, tau, for kinetic activation, represented as percent change from control. Brevenol treated cells demonstrate a 32% change in tau versus vehicle control.
84 8.00E‐04 A Kinetic Inactivation 7.00E‐04 *
) 6.00E‐04 τ ( 5.00E‐04 4.00E‐04 10 nM constant 3.00E‐04 Vehicle
Time 2.00E‐04 1.00E‐04 0.00E+00 Brevenol Brevenal PbTx‐2 Percent change in kinetic inactivation from control
80% B HEK 293 Nav1.4 cells *
60% τ in 40%
20% change
0%
Percent Brevenol Brevenal PbTx‐2 ‐20%
‐40%
*Indicates significant difference
Figure 39: A) Effect of 10 nM brevenol, brevenal and PbTx-2 on the kinetic inactivation of the sodium current in HEK 293 with Nav1.4 cells. Only the PbTx-2 treated cells showed a significant change in inactivation time constant versus vehicle control (Z test = -2.69: p value < 0.008). The value for tau for PbTx-2 treated cells were 563 µs on average, while the control for these cells time constant was 360 µs. Error bars represent + s.e.m. (B) Effect of 10 nM brevenal, brevenol and PbTx-2 on the time constant, tau, for kinetic inactivation, represented as percent change from control. PbTx-2 treated cells demonstrate a 56% change in tau versus vehicle control.
85 Automated Patch Clamp Electrophysiology: HEK 293 (Nav1.4) cells- Voltage- Dependent Inactivation and Recovery from Inactivation
A comparison of the voltage-dependent inactivation was performed using whole
cell patch clamp recordings of HEK 293 (NaV1.4) cells. The protocol for these
experiments involved holding each cell at a potential of -120 mV, prepulsing for 250 ms
with increasing 5 mV steps followed by a 10 ms test pulse of -10 mV. V1/2 values, or the
voltage at which half the channels were inactivated, were determined for the following
treatments: 10 nM brevenal, 10 nM brevenol and 10 nM PbTx-2 (Figure 40). In general,
all treatments caused an inactivation of half of the channels in the cell membrane at more negative voltages than for control and the difference between V1/2 values for all
treatments and controls were found to be significant (Figure 40).
Likewise, the time for recovery from voltage-dependent inactivation was
determined for cells treated with 10 nM brevenal, brevenol or PbTx-2 from experiments
that clamped whole cells at -120 mV and applied a -10 mV test pulse at 1 ms intervals
for 20 sweeps. The data obtained was fit using a 1-exponential curve function to obtain
the time constants for the estimation of the rate of recovery. Cells treated with 10 nM
PbTx-2 exhibited a significant increase in the rate of recovery as compared to the
control (Figure 41). In the same manner, brevenal and brevenol treated cells
demonstrated a significant increase in the rate of recovery as compared to the control
(Figure 41), an indication that the channels treated with brevenal, brevenol and PbTx-2 take longer to recover after inactivation than when the cell was just treated with the vehicle control. A summary of results from all automated patch clamp experiments is listed in Table 5.
86 Voltage Dependent Inactivation HEK 293 Nav1.4
Brevenol Brevenal PbTx‐2
0
‐20 10 nM ‐40 Vehicle
(mV) ‐60
1/2 ‐80 V
‐100 * * * ‐120
*Indicates significant difference
Figure 40: Effect of 10nM brevenol, brevenal and PbTx-2 on voltage dependent inactivation of whole cell patch clamp recordings in HEK 293 Nav1.4 cells. The average V1/2 value for cells (n=4) treated with brevenol -96.7 mV versus -85.8 mV for - the control (Z test = 47.64: p value < 0.008). The average V1/2 value for cells treated with 10 nM brevenal (n=4) was 88.3 mV versus -83.9 mV for the control (Z test = -14.74: p value < 0.008). The average V1/2 value for PbTx-2 (n=4) is -90.3 mV and -80.6 mV for the control (Z test = -37.96: p value < 0.008). The results obtained indicate that inactivation occurs at more negative potentials for channels treated with 10 nM concentrations of brevenol, brevenal and PbTx-2. Error bars represent + s.e.m.
87 Recovery from Inactivation HEK 293 Nav1.4 cells 6.00E‐03
5.00E‐03 * * 4.00E‐03 *
(s) 10 nM 3.00E‐03 Vehicle Time 2.00E‐03
1.00E‐03
0.00E+00 Brevenol Brevenal PbTx‐2
*Indicates significant difference
Figure 41: Effect of 10 nM brevenol, brevenal and PbTx-2 on the voltage dependent recovery from inactivation in HEK 293 Nav1.4 cells. Results indicate a significant change in the rate of recovery from the control for all treatments. The time constant for recovery of 10 nM brevenol (n=4) was 4.82 ms versus 3.04 ms for control treated cells (Z test = 5.07: p value < 0.008). The time constant for recovery of 10 nM brevenal (n=4) was 2.73 ms versus 0.85 ms for control treated cells (Z test = 4.37: p value < 0.008). The time constant for recovery of 10 nM PbTx-2 (n=4) was 4.20 ms versus 1.04 ms for control treated cells (Z test = 4.02: p value < 0.008). Error bars represent + s.e.m.
88 Table 5: Summary of the effects of 10 nM brevenal, 10 nM brevenol and 10nM PbTx-2 on properties of VSSC using N2a cells and HEK 293 transfected with NaV1.4 channels. TABLE 3 Test Cell Type Test Cell Type
Treatment (10 nM) N2a HEK 293 Nav1.4 Treatment (10 nM) N2a HEK 293 Nav1.4 Brevenol ‐ shift ‐ shift* Brevenol less current more current Conductance Brevenal ‐ shift* ‐ shift* Change in IMAX Brevenal more current* more current* PbTx‐2 ‐ shift* ‐ shift* PbTx‐2 less current less current* Brevenol faster * slower* Brevenol + shift* + shift* Kinetic ActivationBrevenal slower* faster Voltage‐dependent Inactivation Brevenal + shift* + shift* PbTx‐2 slower* slower PbTx‐2+ shift* + shift* Brevenol slower slower Brevenol slower slower* Kinetic InactivationBrevenal faster faster Recovery from Inactivation Brevenal slower slower* PbTx‐2 slower* slower* PbTx‐2 slower* slower* *Indicates significant difference; ‐ shift to a more negative potential while a + shift is a shift to a lessnegative potential
89 DISCUSSION
Receptor Binding Studies
Inhibition experiments that demonstrate no competition between other ligands for
VSSC receptors and 3H-brevenol binding provided evidence that the lipid soluble
antitoxin, brevenal, and its alcohol derivative, brevenol, bind with relatively high affinity
to a site on neuronal VSSCs that is distinct. Competitive inhibition of 3H-PbTx-3 binding
by brevenal and brevenol indicated that there are interactions between these ligands
and site 5 of VSSC. A novel semisynthetic probe, 3H-brevenol, was synthesized to
investigate the specific nature of brevenal/ol binding. Competition studies against 3H-
brevenol using ligands specific for five known neurotoxin receptor sites 1-5 and an
ENaC sodium channel blocker site, known for the binding of amiloride, demonstrated
that the brevenal/ol site on VSSC is distinct from these receptor sites. Recently
deltamethrin, a type of pyrethroid which binds to receptor site 7 of VSSCs (Wang and
Wang 2003), has been used in competition studies with 3H-brevenol. Deltamethrin did
not inhibit the binding at the brevenal/ol receptor (personal communication Dr. Henry
Jacocks). Binding at a sixth receptor site, which is characterized by the binding of -
conotoxin (Fainzilber et al. 1994), was not studied in our experiments due to its limited
availability. Future studies could address interactions between -conotoxin and (3H) brevenol.
Interestingly, brevenal/ol did interact with site 5 of VSSC, the brevetoxin (PbTx) binding site. Brevenal/ol reduced radiolabeled 3H-PbTx-3 binding at nanomolar to
picomolar concentrations. However, PbTxs (2 and 3) of backbone type B were unable
to reduce 3H-brevenol binding from its receptor site. The fact that brevetoxin did
90 reduce 3H-brevenol binding indicated that a simple competitive model in which
brevenal/ol and PbTx utilize the same receptor site, did not fit for of the observed
3 binding pattern. In spite of this, the similarity of the Bmax for H-PbTx-3 (7.1 pmol/ mg of
3 synaptosomal protein) and the Bmax for H-brevenol (7.2 pmol/ mg of synaptosomal
protein) provided a clue to explain the complex binding pattern of brevenal/ol on VSSCs
and also implied a 1:1 binding stoichiometry.
Explanations for this binding pattern were dependent on the close proximity of
the brevenal/ol receptor to the brevetoxin site (essentially equal in concentration in
synaptosomes). The process in which ligands bind to receptors follows the kinetics and
fundamental principles of substrates binding to enzymes. Segel proposed five plausible
models of competitive enzyme-substrate interactions (1968) (Figure 42). Using Segel’s
models (which have been adapted to represent receptor binding interactions), the
interaction between the brevetoxin site and brevenal/ol binding can be defined by
incorporating the information obtained from our experiments.
First, model 1 did represent the data observed (Figure 42) because this model
suggested that both PbTxs and brevenal/ol bind to the same receptor sites. The nature
of this model suggested that ligands for this receptor should have structurally similar
binding sites. Brevetoxins and brevenal/ol both are polyethers produced by the marine
dinoflagellate Karenia brevis. However, our competition studies indicated that binding of brevetoxin did not displace the binding of our novel receptor specific ligand, 3H-
brevenol. If brevetoxin and brevenal/ol occupied the same receptor then the presence of
one of these compounds in excess should displace the other. However, our results
indicated that mutual exclusion by both ligands did not occur.
91 The next model, 3, (Figure 42) implied that when a radiolabled ligand like 3H-
PbTx-3 is bound to its receptor site that it shares a common binding site with another receptor, like one which binds 3H-brevenol in our studies. Binding using this type of
competitive inhibition would prevent either ligand from binding when another is bound.
This means that when 3H-PbTx-3 is bound then brevenal and derivatives would be
unable to bind to its receptor and vice versa. This model seemed unlikely to fit the case
of the PbTx and brevenol binding sites because we have seen in competition
experiments that at nanomolar to picomolar concentrations we reduced 3H-PbTx-3
using brevenal and brevenol. However PbTx-2 or 3 were unable to compete with 3H- brevenol for its binding site. Furthermore, this explanation could serve as the reason that model 4 would also be unacceptable as the most plausible explanation of the interactions between our novel receptor site and the brevetoxin (site 5) receptor of
VSSC. Finally, model 5 (Figure 42) did not provide the best explanation of the interactions between these two receptors because it suggested that the binding of brevenal/ol causes a conformational change in the brevetoxin receptor which prevents the binding of brevetoxins. Change in the conformation of the brevetoxin receptor implied a loss in the available receptor sites which results in a reduction in the maximum amount of binding sites (BMax). There was no significant difference in the BMax in our
preparation for the number of receptor sites for PbTx or brevenal/ol. Also, a change in
conformation of the brevetoxin receptor implies non-competitive inhibition. However,
Lineweaver-Burk plots of our results suggested a competitive inhibition of brevetoxin in
the presence of brevenal or brevenol. Finally, our experiments indicated that although
binding of brevenol and brevenal inhibited the binding of 3H-PbTx-3 to its receptor, the
92
Figure 42: (adapted from Segel 1968). Interactions between cell surface receptors, ligands (L) and inhibitors (I). These interactions are used to explain the binding of
PbTxs to site 5 and the novel brevenal/ol receptor located on VSSCs.
93 reverse is not true. In other words, there was no effect on 3H-brevenol binding or no
change in the brevenal/ol receptor when PbTx-2 or PbTx-3 was bound in excess to its
receptor. Therefore, model 2 (Figure 42) provided the best explanation for binding at
our novel receptor and its interaction with the brevetoxin receptor. This model proposed that binding by brevenal and its derivatives sterically hinders the binding of PbTxs from its site. Notice, however that binding of brevetoxin will not prevent the binding of
brevenol in this model.
In current literature, there is evidence that the presence of brevenal inhibits the
physiological effects of brevetoxins. Brevenal has been shown to be an inhibitor of the
neurotoxic effect of ciguatoxin, another known site 5 VSSC ligand (Lombet et al. 1987;
Mattei et al. 2008; Nguyen-Huu et al. 2009). Also, the known physiological activity of brevenal has been shown to be nontoxic and/or protective of the PbTx effect in studies involving fish, DNA damage in human lymphocytes and sheep inhalation studies
(Bourdelais et al. 2004; Sayer et al. 2005; Abraham et al. 2005). Thus, brevenal/ol is the first natural, nontoxic ligand shown to have a novel receptor site on the voltage sensitive (gated) sodium channel. Based on our results, this inhibition occured at the level of the receptor where the brevetoxin receptor is sterically hindered by brevenal preventing the characteristic physiological response resultant of brevetoxin binding.
The interactions between brevenal/ol for its receptor site and site 5 of VSSC suggested that a simple competitive model does not exist. Therefore, binding with brevenal/ol may elucidate the nature of binding with other receptor sites associated with voltage sensitive sodium channels. This brings the number of known receptor sites specific for
VSSC to eight, which includes our novel receptor (Catterall 2005; Wang and Wang
94 2003). Future directions could include isolation and localization of the brevenal/ol
receptor. It is known that the brevetoxin receptor is localized within the fourth domain
between the fifth segment and the first domain sixth transmembrane segment of VSSCs
(Trainer et al., 1991, 1994; Figure 1 In Chapter 1: Introduction). Photoaffinity labeling
studies similar to those performed by Trainer and colleagues could aid in localization of
the brevenal/ol receptor.
Automated Patch Clamp Studies
This work was aimed at determining the effects of brevenal and brevenol on voltage
sensitive sodium channels by focusing on changes in the sodium current (INa) and other
properties of VSSCs. The effect of brevenal/ol on VSSC was investigated by measuring
changes in kinetics, voltage dependent inactivation and recovery from inactivation.
Typically, VSSCs alternate through a series of steps when activated by electrical
impulses (Figure 43). First, VSSCs are activated by a change in membrane voltage
potential which causes the channels to open. From the open configuration, channels
progress to a state of inactivation. During inactivation, VSSCs are temporarily
unavailable for reactivation. Once VSSCs recover from inactivation, they become
available to be activated (original state, closed) once again.
Efforts were focused on examining the effects of brevenal/ol on Nav1.4 channels and
on VSSCs found in a murine neuroblastoma (N2a) cell line. Voltage sensitive sodium
channels in N2a cells were chosen because these cell lines have been used extensively to screen novel compounds for neurotoxic properties (LePage et al. 2005) and because
PbTxs demonstrate increased selectively in neuronal preparations. Likewise, Hek 293
95 CLOSED
INACTIVATED OPEN
Figure 43: The voltage sensitive sodium channel alternates between three states, closed, open and inactivated. The arrows between Inactivated and closed indicate when channels are recovering from inactivation. Channels must recover from inactivation in order to return to their original resting state (closed) be available to be reactivated
96 cells transfected with Nav1.4 sodium channels were used because type B brevetoxins
show increased selectively in binding for the skeletal muscle isoform, Nav1.4 of VSSC
(Dechraoui and Ramsdell 2003). The results of brevenal/ol on both channel types were compared with the actions of a known VSSC receptor ligand for site 5, brevetoxin 2
(PbTx-2).
In N2a cells, only brevenal not brevenol caused a significant negative shift in conductance-voltage (G-V) relationships. This negative shift in conductance correlates to a negative shift in the activation potential. As a result, channels were activated at more negative potentials than normal. However, in Hek 293 NaV1.4 cells, both brevenal and brevenol caused a negative shift in G-V relationships. In both cell lines, changes in G-V relationships are relative to their own internal control. The magnitude of change in conductance by brevenal was higher in N2a cells versus HEK 293 NaV1.4 cells. The percent change from control for cells treated with brevenal in N2a cells was
39% and in HEK 293 NaV1.4 cells was 8%, while the change in G-V relationships for brevenol in HEK 293 NaV1.4 cells was 24%. Thus, treatment with brevenal had a
greater effect on activation, shifting it to more negative voltages in N2a cells while
brevenol only exhibited an effect on the conductance of VSSCs in HEK 293 NaV1.4 cells. This indicated that brevenal shows an increased selectivity for neuronal sodium channels versus channels associated with skeletal muscle. Brevenol, on the other hand, demonstrated significant activity in Nav1.4 sodium channels which are primarily
located in skeletal muscle.
In comparison, PbTx-2 shifted the conductance or activation of sodium channels to
more negative values in both cell lines although its effect is greater in neuronal (N2a)
97 sodium channels (28% change) than in NaV1.4 channels (19% change from control).
The results for PbTx-2 are consistent with results from prior studies. In fact, brevetoxins
(type brevetoxin B or PbTx-2 backbone) have been demonstrated to change the voltage
at which channels are activated in several studies. Most studies indicated that PbTx-3
(type B backbone) caused a shift of the activation curve to more negative potentials
(Huang et al. 1984; Jeglitsch et al. 1998; Purkerson et al. 1999; Sheridan and Alder
1989; Wu et al. 1985). Another study showed that Pbtx-3, PbTx-2, and all derivatives of
brevetoxin, which are active on VSSC, caused a shift in activation to more negative
values (Baden et al. 1996).
Changes in the kinetics of activation and inactivation for sodium currents
produced by neuronal (N2a) and NaV1.4 sodium channels treated with brevenal,
brevenol and PbTx-2 were also examined. N2a cells treated with brevenal exhibited
slower activation kinetics relative to control treatment. This is in contrast to N2a cells
treated with brevenol, which caused the kinetic activation of the channel to become faster. Conversely, like brevenal in N2a cells, brevenol slowed the activation of NaV1.4 channels. Brevenol, therefore, exhibited a difference in effect on kinetic activation in different sodium channel types. In neuronal channels, brevenol accelerated kinetic activation while in VSSCs located in muscle cells it slowed activation of VSSCs. In comparison, PbTx-2 slowed the time at which channels are activated in N2a cells like brevenal but this was opposite of the effect of brevenol in this cell type. This was the first indication that the effects of brevenol and PbTxs on sodium channels may be antagonistic in nature. The differences in the actions of brevenol and brevenal on sodium channel activity may be the result of solubility across the membrane. One
98 reason for these differences is that brevenal, not brevenol, may infiltrate the membrane
similar to PbTx-2 which would account for the similarities in sodium channel response
by this ligand.
While activation describes the opening of VSSCs which allow for the movement
of Na+ ions across a membrane down its concentration gradient, inactivation is
characterized by the closing of an inactivation gate to prevent further sodium ion
conductance, until the membrane reaches its resting potential due to ion pumping
mechanisms to re-establish the gradient. Neither brevenal nor brevenol had a significant
effect on kinetic inactivation in either type of sodium channels. However, PbTx-2
significantly prolonged the time where channels were inactivated both N2a and HEK cell
lines (by 16% and 56%, respectively). To date, this is the first study that reports any
changes in inactivation kinetics by PbTxs.
The final two aspects of the INa that were investigated are voltage dependent
inactivation and recovery from inactivation. Analysis of voltage dependent inactivation is based onV1/2 values, which is the voltage at which half the channels are inactivated.
For all treatments (brevenal/ol and PbTx-2), results indicated that channels inactivate at more negative potentials versus control cells. Recovery from inactivation is a property of sodium channels which describes the time constant needed for VSSC to go from an inactivated state to a resting state in which they are available for subsequent activation.
Brevenol and brevenal slowed the time for recovery from inactivation in NaV1.4 sodium
channels only. These ligands had no significant effect on neuronal sodium channels at
this concentration (10 nM). Future experiments should explore the concentration
99 dependence of brevenal/ol activity. In contrast, PbTx-2 slowed recovery from inactivation in both channel types, neuronal and muscular.
The actions of any compound on voltage-dependent inactivation and recovery from inactivation in NaV1.4 channels are important because VSSCs in muscle cells function in the transmission of nerve impulses at the neuromuscular junction. For a cell to fire a number of action potentials, channels must quickly recover from the inactivated state to remain available for subsequent activation. This conduction of action potentials represents a fundamental means of communication between muscles and the nervous system. “Slowed” inactivation along with the ability of the channel to recover from inactivation reduces cell excitability which may result in eventual termination of the cell.
Future studies should test the response of VSSCs in the NaV1.4 channel type and N2a cells to a concentration of brevenal/ol closer to the KD obtained in receptor binding
experiments since this parameter represents the affinity of brevenal/ol for its receptor.
Also, studies which explore combinations of brevetoxin against brevenal/ol to
investigate any potential protective or inhibitory effects at this level should be
performed.
100 CONCLUSIONS
Receptor Binding
3 Brevenol, an alcohol derivative, and a new radioligand, H-brevenol (KD = 68
nM), are a valuable ligands in the characterization of binding to a novel target in
excitable tissues.
The novel receptor site (Bmax = 7.2 pmol/mg of protein) which binds brevenal and
brevenol (KI = 75 nM and 57 nM, respectively) interacts with the brevetoxin
binding site associated with voltage sensitive sodium channels.
The interaction between brevenal/ol and brevetoxin is not a simple pattern of
mutual competitive inhibition. Data indicated that brevenal/ol can eliminate the
binding of brevetoxin at site 5 (KI = 98 nM and 661 nM for brevenal and brevenol,
respectively), but the reciprocal is not true.
3H-Brevenol is an important probe in characterizing compounds which target
VSSCs.
Electrophysiology of N2a and HEK 293 NaV1.4 cells
In planar patch clamp electrophysiology studies: All compounds (brevenal,
brevenol and PbTx-2) cause a positive shift in the voltage dependent inactivation
and slow down the recovery from inactivation.
Brevenol increases the activation kinetics of sodium channels in neuroblastoma
(N2a) cells, while PbTx-2 decreases the activation kinetics.
101 The magnitude of the brevenal/ol effect on sodium channels depends on the type
of channel. Brevenal and PbTx-2 exhibit a greater effect on VSSCs in N2a cells
than the muscle (NaV1.4) sodium channel isoforms. This is not true for brevenol.
102 REFERENCES
1. Abraham, W. M., Bourdelais, A. J., Sabater, J. R., Ahmed, A., Lee, T. A., Serebriakov, I., and Baden, D. G. (2005) Airway responses to in an animal model. American Journal of Respiratory and Critical Care Medicine 171(1), 26-34 2. Aidley, D. J., Stanfield, P.R. (1996) Ion Channels: Molecules in Action, Cambridge University Press, New York, NY 3. Aidley, D. J. (1998) The Physiology of Excitable Cells, 4th Ed., Cambridge University Press, New York, NY 4. Anger, T., Madge, D. J., Mulla, M., and Riddall, D. (2001) Medicinal chemistry of neuronal voltage-gated sodium channel blockers. Journal of Medicinal Chemistry 44(2), 115-137 5. Armstrong, C. M. (1981) Sodium channels and gating currents. Physiol Rev 61, 644-683 6. Baden D.G., R. K. S., Gawley R.E., Jeglitsch G., Adams D.J. (1996) Marine toxins: scientific approaches, synthetic transformations, and molecular mechanisms. In: Yasumoto, T., Oshima, Y., Fukuyo, Y. (ed). Harmful and Toxic Algal Blooms Intergovernmental Oceanographic Commission of UNESCO, Paris 7. Baden, D. G. (1983) Marine food-borne dinoflagellate toxins.International Review of Cytology 82, 99-150 8. Baden, D. G. (1989) Brevetoxins: unique polyether dinoflagellate toxins. Faseb J 3(7), 1807-1817 9. Baden, D. G., Fleming, L. E., Bean, J. A. (1995) Marine Toxins. In: deWolf, F. A. (ed). Handbook of Clinical Neurology: Intoxification of the Nervous System Part H. Natural Toxins and Drugs Elsevier Press 10. Baden, D. G., Rein, K.S., Gawley, R.E.,. (1998) Marine Toxins: How They Are Studied and What They Can Tell Us. In: Cooksey, K. E. (ed). Molecular Approaches to the Study of the Ocean, Chapman & Hall, London 11. Baden, D. G., Melinek, R., Sechet, V., Trainer, V. L., Schultz, D. R., Rein, K. S., Tomas, C. R., Delgado, J., and Hale, L. (1995) Modified immunoassays for polyether toxins: implications of biological matrixes, metabolic states, and epitope recognition. J AOAC Int 78(2), 499-508 12. Baden, D. G., and Mende, T. J. (1982) Toxicity of 2 Toxins from the Florida Red Tide Marine Dinoflagellate Ptychodiscus brevis. Toxicon 20(2), 457-462 13. Baden, D. G., Rein, K. S., Gawley, R. E., Jeglitsch, G., and Adams, D. J. (1994) Is the A-ring lactone of brevetoxin PbTX-3 required for sodium channel orphan receptor binding and activity? Natural Toxins 2(4), 212-221 14. Bagnis, R., Chanteau, S., Chungue, E., Hurtel, J. M., Yasumoto, T., and Inoue, A. (1980) Origins of ciguatera fish poisoning: a new dinoflagellate, Gambierdiscus toxicus Adachi and Fukuyo, definitively involved as a causal agent. Toxicon 18(2), 199-208 15. Barchi, R. L. (1995) Molecular pathology of the skeletal muscle sodium channel. In. Annual Review of Physiology 16. Barnola, F. V., Villegas, R., and Camejo, G. (1973) Tetrodotoxin receptors in plasma membranes isolated from lobster nerve fibers. Biochimica et biophysica acta 298(1), 84-94
103 17. Benforado, J. M. (1967) The veratrum alkaloids. In: Root, W. S., Hofmann F. G. (ed). Physiological Pharmacology, Academic Press, New York 18. Berg, J. M., Tymoczko, J. L., Stryer, L. (2002) Biochemistry, Fifth Ed., W H Freeman and Company, New York, NY 19. Berman, F. W., and Murray, T. F. (1999) Brevetoxins cause acute excitotoxicity in primary cultures of rat cerebellar granule neurons. Journal of Pharmacology and Experimental Therapeutics 290(1), 439-444 20. Bourdelais, A. J., Campbell, S., Jacocks, H., Naar, J., Wright, J. L. C., Carsi, J., and Baden, D. G. (2004) Brevenal is a natural inhibitor of brevetoxin action in sodium channel receptor binding assays. Cellular and Molecular Neurobiology 24(4), 553-563 21. Bourdelais, A. J., Jacocks, H. M., Wright, J. L., Bigwarfe, P. M., Jr., and Baden, D. G. (2005) A new polyether ladder compound produced by the dinoflagellate Karenia brevis. Journal of Natural Products 68(1), 2-6 22. Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72(1-2), 248-254 23. Brown, G. B. (1986) Tritiated batrachotoxin a benzoate binding to voltage- sensitive sodium channels inhibition by the channel blockers tetrodotoxin and saxitoxin. Journal of Neuroscience 6(7), 2064-2070 24. Brown, G. B., Tieszen, S. C., Daly, J. W., Warnick, J. E., and Albuquerque, E. X. (1981) Batrachotoxinin-A 20-alpha-benzoate: a new radioactive ligand for voltage sensitive sodium channels. Cell Mol Neurobiol 1(1), 19-40 25. Cannon, S. C. (1996) Sodium channel defects in myotonia and periodic paralysis. In. Annual Review of Neuroscience 26. Catterall, W. A. (1977) Activation of the action potential Na+ ionophore by neurotoxins: An allosteric model. Journal of Biological Chemistry 252(23), 8669- 8676 27. Catterall, W. A. (1979) Neurotoxins as allosteric modifiers of action potential sodium channels. Toxicon 17(SUPPL. 1), 1979 28. Catterall, W. A. (1980) Neurotoxins that act on voltage sensitive sodium channels in excitable membranes In. George, R. and R. Okun 29. Catterall, W. A. (1981) Inhibition of voltage sensitive sodium channels in neuroblastoma cells by antiarrhythmic drugs. Molecular Pharmacology 20(2), 356-362 30. Catterall, W. A. (1984) The molecular basis of neuronal excitability. Science 223(4637), 653-661 31. Catterall, W. A. (1986) Voltage-dependent gating of sodium channels correlating structure and function. Trends in Neurosciences 9(1), 7-10 32. Catterall, W. A., and Beress, L. (1978) Sea anemone toxin and scorpion toxin share a common receptor site associated with the action potential sodium ionophore. Journal of Biological Chemistry 253(20), 7393-7396 33. Catterall, W. A., and Coppersmith, J. (1981) High affinity saxitoxin receptor sites in vertebrate heart evidence for site associated with autonomic nerve endings. Molecular Pharmacology 20(3), 526-532
104 34. Catterall, W. A., and Coppersmith, J. (1981) Pharmacological properties of sodium channels in cultured rat heart cells. Molecular Pharmacology 20(3), 533- 542 35. Catterall, W. A., and Gainer, M. (1985) Interaction of brevetoxin A with a new receptor site on the sodium channel. Toxicon 23(3), 497-504 36. Catterall, W. A., Hartshorne, R. P., and Beneski, D. A. (1982) Molecular properties of neurotoxin receptor sites associated with sodium channels from mammalian brain. Toxicon 20(1), 27-40 37. Catterall, W. A., and Morrow, C. S. (1978) Binding to saxitoxin to electrically excitable neuroblastoma cells. Proc Natl Acad Sci USA 75(1), 218-222 38. Catterall, W. A., Morrow, C. S., Daly, J. W., and Brown, G. B. (1981) Binding of batrachotoxinin a 20-alpha benzoate to a receptor site associated with sodium channels in synaptic nerve ending particles. Journal of Biological Chemistry 256(17), 8922-8927 39. Catterall, W. A., Morrow, C. S., and Hartshorne, R. P. (1979) Neurotoxin binding to receptor sites associated with voltage sensitive sodium channels in intact lysed and detergent solubilized brain membranes. Journal of Biological Chemistry 254(22), 11379-11387 40. Catterall, W. A., Perez-Reyes, E., Snutch, T. P., and Striessnig, J. (2005) International Union of Pharmacology. XLVIII. Nomenclature and structure- function relationships of voltage-gated calcium channels. Pharmacological Reviews 57(4), 411-425 41. Catterall, W. A., and Risk, M. (1981) Toxin T-46 from Ptychodiscus brevis formerly Gymnodinium breve enhances activation of voltage sensitive sodium channels by veratridine. Molecular Pharmacology 19(2), 345-348 42. Cestele, S., and Catterall, W. A. (2000) Molecular mechanisms of neurotoxin action on voltage-gated sodium channels. Biochimie 82(9-10), 883-892 43. Cestele, S., and Gordon, D. (1998) Depolarization differentially affects allosteric modulation by neurotoxins of scorpion alpha-toxin binding on voltage-gated sodium channels. Journal of Neurochemistry 70(3), 1217-1226 44. Cestele, S., Khalifa, R. B., Pelhate, M., Rochat, H., and Gordon, D. (1995) Alpha- Scorpion Toxins Binding on Rat Brain and Insect Sodium Channels Reveal Divergent Allosteric Modulations by Brevetoxin and Veratridine. Journal of Biological Chemistry 270(25), 15153-15161 45. Cestele, S., Khalifa, R. B., Pelhate, M., Rochat, H., and Gordon, D. (1996) Alpha scorpion toxins binding on rat brain and insect sodium channels reveal divergent allosteric modulations by brevetoxin and veratridine. Toxicon 34(3), 303-304 46. Cestele, S., Sampieri, F., Rochat, H., and Gordon, D. (1996) Tetrodotoxin reverses brevetoxin allosteric inhibition of scorpion alpha-toxin binding on rat brain sodium channels. Journal of Biological Chemistry 271(31), 18329-18332 47. Chahine, M., Sirois, J., Marcotte, P., Chen, L. Q., and Kallen, R. G. (1998) Extrapore residues of the S5-S6 loop of domain 2 of the voltage-gated skeletal muscle sodium channel (rSkM1) contribute to the mu-conotoxin GIIIA binding site. Biophysical Journal 75(1), 236-246 48. Chicheportiche, R., Balerna, M., Lombet, A., Romey, G., and Lazdunski, M. (1980) Synthesis of new highly radioactive tetrodotoxin derivatives and their
105 binding properties to the sodium channel. European Journal of Biochemistry 104(2), 617-625 49. Chou, H. N., and Shimizu, Y. (1982) A new polyether toxin from Gymnodinium breve. Tetrahedron Letters 23(52), 5521-5524 50. Colquhoun, D., Henderson, R., and Ritchie, J. M. (1972) The binding of labelled tetrodotoxin to non-myelinated nerve fibres. The Journal of Physiology 227(1), 95-126 51. Cruz, L. J., Gray, W. R., Olivera, B. M., Zeikus, R. D., Kerr, L., Yoshikami, D., and Moczydlowski, E. (1985) Conus geographus toxins that discriminate between neuronal and muscle sodium channels. Journal of Biological Chemistry 260(16), 9280-9288 52. Cruz, L. J., Kupryszewski, G., Lecheminant, G. W., Gray, W. R., Olivera, B. M., and Rivier, J. (1989) Mu conotoxin GIIIA a peptide ligand for muscle sodium channels chemical synthesis radiolabeling and receptor characterization. Biochemistry 28(8), 3437-3442 53. Daly, J. W., and Witkop, B. (1971) Chemistry and pharmacology of frog venoms In. Buecherl, Wolfgang and Eleanor E. Buckley 54. de Weille, J. R., Brown, L. D., and Narahashi, T. (1990) Pyrethriod modifications of the activation and inactivation kinetics of the sodium channels in squid giant axons. Brain Research 512(1), 26-32 55. Dodd, P. R., Hardy, J. A., Oakley, A. E., Edwardson, J. A., Perry, E. K., and Delaunoy, J. P. (1981) A rapid method for preparing synaptosomes comparison with alternative procedures. Brain Research 226(1-2), 107-108 56. Donaldson, S. H., and Boucher, R. C. (2007) Sodium channels and cystic fibrosis. Chest 132(5), 1631-1636 57. Donaldson, S. H., Hirsh, A., Li, D. C., Holloway, G., Chao, J., Boucher, R. C., and Gabriel, S. E. (2002) Regulation of the epithelial sodium channel by serine proteases in human airways. Journal of Biological Chemistry 277(10), 8338-8345 58. Fainzilber, M., Gordon, D., Hasson, A., Spira, M. E., and Zlotkin, E. (1991) Mollusc-specific toxins from the venom of Conus textile neovicarius. European Journal of Biochemistry / FEBS 202(2), 589-595 59. Fainzilber, M., Kofman, O., Zlotkin, E., and Gordon, D. (1994) A new neurotoxin receptor site on Sodium channels is iIdentified by a conotoxin that affects sodium channel inactivation in molluscs and acts as an antagonist in rat brain. Journal of Biological Chemistry 269(4), 2574-2580 60. Fainzilber, M., Lodder, J. C., Kits, K. S., Kofman, O., Vinnitsky, I., Van Rietschoten, J., Zlotkin, E., and Gordon, D. (1995) A new conotoxin affecting sodium current inactivation interacts with the delta-conotoxin receptor site. Journal of Biological Chemistry 270(3), 1123-1129 61. Farre, C., Stoelzle, S., Haarmann, C., George, M., Briiggemann, A., Fertig, N. (2007) Automated ion channel screening: patch clamping made easy. Expert Opin Ther Targets 11(2), 557-565 62. Goldin, A. L. (2002) Evolution of voltage-gated Na+ channels. J Exp Biol 205(5), 575-584 63. Gonoi, T., Ashida, K., Feller, D., Schmidt, J., Fujiwara, M., and Cattrall, W. A. (1986) Mechanism of action of a polypeptide neurotoxin from the coral Goniopora
106 on sodium channels in mouse neuroblastoma cells. Molecular Pharmacology 29(4), 347-354 64. Gonoi, T., Ohizumi, Y., Kobayashi, J., Nakamura, H., and Catterall, W. A. (1987) Action of a polypepetide toxin fromthe marine snal Conus striatus on voltage- sensitive sodium channels. Molecular Pharmacology 32(5), 691-698 65. Gordon, D. (1998) Neurotoxin receptor sites on sodium channels-who interacts with who and how? Toxicon 36(9), 1233-1234 66. Gordon, D., and Cestele, S. (1997) On the allosteric modulation of neurotoxin binding on voltage-gated sodium channels. Biophysical Journal 72(2), A4 67. Gordon, D., Cestele, S., Sampieri, F., and Rochat, H. (1996) Unexpected allosteric interactions between TTX and brevetoxin receptor sites on Na channels. Progress in Biophysics and Molecular Biology 65(SUPPL. 1), 106 68. Gordon, D., Martin-Eauclaire, M.-F., Cestele, S., Kopeyan, C., Carlier, E., Khalifa, R. B., Pelhate, M., and Rochat, H. (1996) Scorpion toxins affecting sodium current inactivation bind to distinct homologous receptor sites on rat brain and insect sodium channels. Journal of Biological Chemistry 271(14), 8034-8045 69. Graham, A., Hasani, A., Alton, E. W. F. W., Martin, G. P., Marriott, C., Hodson, M. E., Clarke, S. W., and Geddes, D. M. (1993) No added benefit from nebulized amiloride in patients with cystic fibrosis. European Respiratory Journal 6(9), 1243-1248 70. Henderson, R., Ritchie, J. M., and Strichartz, G. R. (1973) The binding of labelled saxitoxin to mammalian non-myelinated nerve fibres. The Journal of Physiology 232(1), 53P-54P 71. Hirsh, A. J., Sabater, J. R., Zamurs, A., Smith, R. T., Paradiso, A. M., Hopkins, S., Abraham, W. M., and Boucher, R. C. (2004) Evaluation of second generation amiloride analogs as therapy for cystic fibrosis lung disease. The Journal of Pharmacology and Experimental Therapeutics 311(3), 929-938 72. Hodgkin, A. L., and Huxley, A. F. (1952) Propagation of electrical signals along giant nerve fibers. Proceedings of the Royal Society of London Series B, Containing papers of a Biological character 140(899), 177-183 73. Hoffman, E. P., Lehmann-Horn, F., and Ruedel, R. (1995) Overexcited or inactive: Ion channels in muscle disease.Cell 80(5), 681-686 74. Huang J. M. C., W., C. H., Baden D. G. (1984) Depolarizing action of a red-tide dinoflagellate brevetoxin on axonal membranes. The Journal of Pharmacology and experimental therapeutics 229, 615-621 75. Isom, L. L. (2001) Sodium channel beta subunits: anything but auxiliary. Neuroscientist 7(1), 42-54 76. Jacques, Y., Fosset, M., and Lazdunski, M. (1978) Molecular properties of the action potential Na+ ionophore in neuroblastoma cells. Interactions with neurotoxins. Journal of Biological Chemistry 253(20), 7383-7392 77. Jeglitsch, G., Rein, K., Baden, D. G., and Adams, D. J. (1998) Brevetoxin-3 (PbTx-3) and its derivatives modulate single tetrodotoxin-sensitive sodium channels in rat sensory neurons. Journal of Pharmacology and Experimental Therapeutics 284(2), 516-525
107 78. Jover, E., Martin-Moutot, N., Couraud, F., and Rochat, H. (1980) Binding of scorpion toxins to rat brain synaptosomal fraction. Effects of membrane potential, ions, and other neurotoxins. Biochemistry 19(3), 463-467 79. Keynes, R. D. (1994) The kinetics of voltage-gated ion channels. Quarterly Reviews of Biophysics 27(4), 339-434 80. Knowles, M. R., Church, N. L., Waltner, W. E., Yankaskas, J. R., Gilligan, P., King, M., Edwards, L. J., Helms, R. W., and Bourcher, R. C. (1990) A pilot study of aerosolized amiloride fro the treatment of lung disease in cystic fibrosis. New England Journal of Medicine 322(17), 1189-1194 81. Kohler, D., App, E., Schmitz-Schumann, M., Wurtemberger, G., and Matthys, H. (1986) Inhalation of amiloride improves the mucociliary and the cough clearance in patients with cystic fibrosis. European journal of respiratory diseases 146, 319- 326 82. Krueger, B. K., and Blaustein, M. P. (1980) Regulation of neuronal sodium channels by neurotoxins In. Shamoo, a. E. 83. Krueger, B. K., Ratzlaff, R. W., Strichartz, G. R., and Blaustein, M. P. (1979) Saxitoxin binding to synaptosomes membranes and solubilized binding sites from rat brain. Journal of Membrane Biology 50(3-4), 287-310 84. Krueger, B. K., Worley, J. F., III, and French, R. J. (1983) Sodium channels from rat brain in planar lipid bilayers. Biophysical Journal 41(2 PART 2), 142A 85. Lawrence, J. C., and Catterall, W. A. (1981) Tetrodotoxin insensitive sodium channels binding of polypeptide neurotoxins in primary cultures of rat muscle cells. Journal of Biological Chemistry 256(12), 6223-6229 86. LePage, K. T., Rainier, J. D., Johnson, H. W., Baden, D. G., and Murray, T. F. (2007) Gambierol acts as a functional antagonist of neurotoxin site 5 on voltage- gated sodium channels in cerebellar granule neurons. The Journal of Pharmacology and Experimental Therapeutics 323(1), 174-179 87. Levinson, S. R., Karlin, A., Kaback, R., Keynes, R. D., and Stevens, C. T. (1986) Future directions in sodium channel research. Annals of the New York Academy of Sciences 479, 439-445 88. Li, W. I., Berman, F. W., Okino, T., Yokokawa, F., Shioiri, T., Gerwick, W. H., and Murray, T. F. (2001) Antillatoxin is a marine cyanobacterial toxin that potently activates voltage-gated sodium channels. Proceedings of the National Academy of Sciences USA 98(13), 7599-7604 89. Lin, Y. Y., Risk, M., Ray, S. M., Van Engen, D., Clardy, J., Golik, J., James, J. C., and Nakanishi, K. (1981) Isolation and structure of brevetoxin B from the red tide dinoflagellate Ptychodiscus brevis Gymnodinium breve Journal of the American Chemical Society 103(22), 6773-6775 90. Lineweaver, H., Burk, D. (1934) The determination of enzyme dissociation constants. Journal of the American Chemical Society 56, 658-666 91. Lombet, A., Bidard, J. N., and Lazdunski, M. (1987) Ciguatoxin and brevetoxins share a common receptor site on the neuronal voltage-dependent Na+ channel. FEBS Lett 219(2), 355-359 92. Lombet, A., Mourre, C., and Lazdunski, M. (1988) Interaction of insecticides of the pyrethroid family with specific binding sites on the voltage-dependent sodium channels from the mammalian brain. Brain Research 459(1), 44-53
108 93. Mattei, C., Wen, P. J., Truong, D., Nguyen-Huu, T. D., Alvarez, M., Benoit, E., Bourdelais, A. J., Lewis, R.J., Baden, D. G., Molgó, J., Meunier, F. A. (2008) Brevenal Inhibits pacific ciguatoxin-1B induced neurosecretion from bovine chromaffin cells. PLoS ONE 3(10), e3448 94. Mattei, C., Dechraoui, M.-Y., Molgo, J., Meunier, F. A., Legrand, A.-M., and Benoit, E. (1999) Neurotoxins targetting receptor site 5 of voltage-dependent sodium channels increase the nodal volume of myelinated axons. Journal of Neuroscience Research 55(6), 666-673 95. Mattei, C., Molgo, J., Legrand, A.-M., and Benoit, E. (1999) Ciguatoxins and brevetoxins: Dissection of their neurobiological actions. Journal de la Societe de Biologie 193(3), 329-344 96. McIntosh, M., Cruz, L. J., Hunkapiller, M. W., Gray, W. R., and Olivera, B. M. (1982) Isolation and strucutre of a peptide tocin from the marine snail Conus magnus. Archives of Biochemistry and Biophysics 218(1), 329-334 97. Molleman, A. (2003) Patch clamping: an introductory guide to patch clamp electrophysiology, John Wiley & Sons, Hoboken, NJ 98. Mullin, M. J., and Hunt, W. A. (1987) Astions of ethanol on voltage-sensitive sodium channels effects on neurotoxin binding. Journal of Pharmacology and Experimental Therapeutics 242(2), 536-540 99. Murrell, R. N., and Gibson, J. E. (2009) Brevetoxins 2, 3, 6, and 9 show variability in potency and cause significant induction of DNA damage and apoptosis in Jurkat E6-1 cells. Archives of Toxicology 83(11), 1009-1019 100. Narahashi, T. (1990) Pharmacology of ion channels: past, present and future. European Journal of Pharmacology 183(1), 149-149 101. Narahashi, T., and Yamasaki, T. (1960) Behaviors of membrane potential in the cockroach giant axons poisoned by DDT. Journal of Cellular and Comparative Physiology 55, 131-142 102. Nguyen-Huu, T. D., Mattei, C., Wen, P. J., Bourdelais, A. J., Lewis, R. J., Benoit, E., Baden, D. G., Molgo, J., and Meunier, F. A. Ciguatoxin-induced catecholamine secretion in bovine chromaffin cells: mechanism of action and reversible inhibition by brevenal. Toxicon 56(5), 792-796 103. Noda, M., Shimizu, S., Tanabe, T., Takai, T., Kayano, T., Ikeda, T., Takahashi, H., Nakayama, H., Kanaoka, Y., and et al. (1984) Primary structure of Electrophorus electricus sodium channel deduced from complementatry DNA sequence. Nature (London) 312(5990), 121-127 104. Noda, M., Suzuki, H., Numa, S., and Stuehmer, W. (1989) A single point mutation confers tetrodotoxin and saxitoxin insensitivity on the sodium channel II. Febs Letters 259(1), 213-216 105. Poli, M. A. (December1985) Characterization of the binding of the Ptychodiscus brevis neurotoxin T17 to sodium channels in rat brain synaptosomes. In., University of Miami, Coral Gables, FL 106. Poli, M. A., Mende, T. J., and Baden, D. G. (1986) Brevetoxins unique activators of voltage-sensitive sodium channels bind to specific sites in rat brain synaptosomes. Molecular Pharmacology 30(2), 129-135
109 107. Purkerson, S. L., Baden, D. G., and Fieber, L. A. (1999) Brevetoxin modulates neuronal sodium channels in two cell lines derived from rat brain. Neurotoxicology 20(6), 909-920 108. Purkerson-Parker, S. L., Fieber, L. A., Rein, K. S., Podona, T., and Baden, D. G. (2000) Brevetoxin derivatives that inhibit toxin activity. Chemistry and Biology (London) 7(6), 385-393 109. Ray, R., and Catterall, W. A. (1978) Membrane potential dependent binding of scorpion toxin to the action potential sodium ionophore. Studies with a 3-(4- hydroxy 3-[125I] iodophenyl) propionyl derivative. J Neurochem 31(2), 397-407 110. Ray, R., Morrow, C. S., and Catterall, W. A. (1978) Binding of the scorpion toxin to receptor sites associated with volate sensitive sodium channels in synaptic nerve ending particles. Journal of Biological Chemistry 253(20), 7307-7313 111. Rein, K. S., Lynn, B., Gawley, R. E., and Baden, D. G. (1994) Brevetoxin B: Chemical modifications, synaptosome binding, toxicity, and an unexpected conformational effect. Journal of Organic Chemistry 59(8), 2107-2113 112. Richie, J. M., and Rogart, R. B. (1977) The binding of labeled saxitoxin tothe sodium channels in normal and denervated mammalian muscle and in amphibian muscle. Journal of Physiology (Cambridge) 269(2), 341-354 113. Riegel, B., Stanger, D. W., and et al. (1949) Paralytic shellfish poison, the primary source of the poison, the marine plankton organism, Gonyaulax catenella. Journal of Biological Chemistry 177(1), 7-11 114. Ritchie, J. M., and Rogart, R. B. (1977) The binding of saxitoxin and tetrodotoxin to excitable tissue In. Adrian, R. H. Et Al. 115. Ritchie, J. M., Rogart, R. B., and Strichartz, G. R. (1976) A new method for labeling saxitoxin and its binding to nonmyelinated fibers tof the rabbit vagus lobster walking leg and garfish olfactory nerves. Journal of Physiology (Cambridge) 261(2), 477-494 116. Satake, M., Murata, M., and Yasumoto, T. (1993) The structure of CTX3C, a ciguatoxin congener isolated from cultured Gambierdiscus toxicus. Tetrahedron Letters 34(12), 1975-1978 117. Sayer, A., Hu, Q., Bourdelais, A. J., Baden, D. G., and Gibson, J. E. (2005) The effect of brevenal on brevetoxin-induced DNA damage in human lymphocytes. Arch Toxicol 79(11), 683-688 118. Sayer, A. N., Hu, Q., Bourdelais, A. J., Baden, D. G., and Gibson, J. E. (2006) The inhibition of CHO-K1-BH4 cell proliferation and induction of chromosomal aberrations by brevetoxins in vitro. Food Chem Toxicol 44(7), 1082-1091 119. Scatchard, G. (1949) The attraction of proteins for small molecules and ions. Annuals of the New York Academy of Sciences 51, 660-672 120. Segel, I. H. (1968) Biochemical Calculations, Second Ed., John Wiley & Sons, New York, NY 121. Sharkey, R. G., Jover, E., Couraud, F., Baden, D. G., and Catterall, W. A. (1987) Allosteric modulation of neurotoxin binding to voltage-sensitive sodium channels by Ptychodiscus brevis toxin 2. Molecular Pharmacology 31(3), 273-278 122. Sheridan, R. E., Alder, M. (1989) The actions of the red tide toxin from Ptychodiscus brevis on single sodium channels in mammalian neuroblastoma cells. Febs Letters 247(2), 448-452
110 123. Shichor, I., Fainzilber, M., Pelhate, M., Malecot, C. O., Zlotkin, E., and Gordon, D. (1996) Interactions of delta-conotoxins with alkaloid neurotoxins reveal differences between the silent and effective binding sites on voltage-sensitive sodium channels. Journal of Neurochemistry 67(6), 2451-2460 124. Shimizu, Y., Chou, H. N., Bando, H., Van Duyne, G., and Clardy, J. C. (1986) Structure of brevetoxin A GB-1 toxin. The most potent toxin in the Florida USA red tide organism Gymnodinium breve Ptychodiscus brevis. Journal of the American Chemical Society 108(3), 514-515 125. Soderlund, D. M., Clark, J. M., Sheets, L. P., Mullin, L. S., Piccirillo, V. J., Sargent, D., Stevens, J. T., and Weiner, M. L. (2002) Mechanisms of pyrethroid neurotoxicity: Implications for cumulative risk assessment. Toxicology 171(1), 3- 59 126. Sommer, H., Meyer, K. F. (1937) Parallytic Shellfish Poisoning. Arch Path 560- 598 127. Strichartz, G. (1982) Structure of the saxitoxin binding site at sodium channels in nerve membranes exchange of tritium bound toxin molecules S. Molecular Pharmacology 21(2), 343-350 128. Strichartz, G. R. (1981) Pharmacological properties of sodium channels in nerve membranes In. Waxman, S. G. and J. M. Ritchie 129. Stuhmer, W., Conti, F., Suzuki, H., Wang, X. D., Noda, M., Yahagi, N., Kubo, H., and Numa, S. (1989) Structural parts involved in activation and inactivation of the sodium channel. Nature 339(6226), 597-603 130. Tamkun, M. M., and Catterall, W. A. (1981) Reconstitution of the voltage sensitive sodium channel of rat brain from solubilized components. Journal of Biological Chemistry 256(22), 11457-11463 131. Thomsen, W. J., and Catterall, W. A. (1989) Localization of the receptor site for alpha scorpion toxins by antibody mapping implications from sodium channel topology Proceedings of the National Academy of Sciences of the United States of America 86(24), 10161-10165 132. Tong, Z., Illek, B., Bhagwandin, V. J., Verghese, G. M., and Caughey, G. H. (2004) Prostasin, a membrane-anchored serine peptidase, regulates sodium currents in JME/CF15 cells, a cystic fibrosis airway epithelial cell line. American Journal of Physiology - Lung Cellular and Molecular Physiology 287(5), L928- L935 133. Trainer, V. L., Baden, D. G., and Catterall, W. A. (1994) Identification of peptide components of the brevetoxin receptor site of rat brain sodium channels. Journal of Biological Chemistry 269(31), 19904-19909 134. Trainer, V. L., Baden, D. G., and Catterall, W. A. (1995) Detection of marine toxins using reconstituted sodium channels. Journal of AOAC International 78(2), 570-573 135. Trainer, V. L., Brown, G. B., and Catterall, W. A. (1996) Site of covalent labeling by a photoreactive batrachotoxin derivative near transmembrane segment IS6 of the sodium channel alpha subunit. Journal of Biological Chemistry 271(19), 11261-11267
111 136. Trainer, V. L., Edwards, R. A., Szmant, A. M., Stuart, A. M., Mende, T. J., and Baden, D. G. (1990) Brevetoxins unique activators of voltage-sensitive sodium channels In. Hall, S. and G. Strichartz 137. Trainer, V. L., Moreau, E., Guedin, D., Baden, D. G., and Catterall, W. A. (1993) Neurotoxin binding and allosteric modulation at receptor sites 2 and 5 on purified and reconstituted rat brain sodium channels. The Journal of Biological Chemistry 268(23), 17114-17119 138. Trainer, V. L., Thomsen, W. J., Catterall, W. A., and Baden, D. G. (1991) Photoaffinity labeling of the brevetoxin receptor on sodium channels in rat brain synaptosomes. Molecular Pharmacology 40(6), 988-994 139. Ulbricht, W. (1979) Drugs that predominantly affect sodium channels. Naunyn- Schmiedeberg's Archives of Pharmacology 803(SUPPL), R1 140. Vincent, J. P., Balerna, M., Barhanin, J., Fosset, M., and Lazdunski, M. (1980) Binding of sea anemone toxin to receptor sites associated with gating system of sodium channel in synaptic nerve endings in vitro. Proc Natl Acad Sci USA 77(3), 1646-1650 141. Wagner, H. H., and Ulbricht, W. (1976) Saxitoxin and procaine act independently on seperate sites of the sodium channel. Pfluegers Archiv European Journal of Physiology 364(1), 65-70 142. Walz, W. (2007) Patch-Clamp Techniques, Second Ed., Humana Press, Totowa, NJ 143. Wang, S.-Y., and Wang, G. K. (2003) Voltage-gated sodium channels as primary targets of diverse lipid-soluble neurotoxins. Cellular Signalling 15(2), 151-159 144. Willow, M., and Catterall, W. A. (1982) Inhibition of binding of tritium labeled batrachotoxinin A-20-alpha benzoate to sodium channels by the anticonvulsant drugs diphenylhydantoin and carbamazephine. Molecular Pharmacology 22(3), 627-635 145. Wu, C. H., Huang, J.M.C., Vogel, S.M., Luke, V.S., Atchinson, W.D., Narahashi, T. (1985) Actions of Ptychodiscus brevis toxins on nerve and muscle membranes. Toxicon 23, 481-487 146. Yamamura, H. I., Enna, S. J., and Kuhar, M. J. (1985) Neurotransmitter receptor binding 2ND Edition
112