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Biomedical subjects

G R Strichartz

Publications and source records attributed to G R Strichartz.

At least 91 records · Page 5Linked to original sources
PubMed1980

Saxitoxin binding to sodium channels in head extracts from wild-type and tetrodotoxin-sensitive strains of Drosophila melanogaster.

Extracts prepared from heads of Drosophila melanogaster show high-affinity binding (KD = 1.9 nM) of [3H]saxitonin, a compound known to bind to and block voltage-sensitive sodium channels in other organisms. The interaction between saxitoxin and the Drosophila saxitoxin receptor is non-cooperative and reversible with a half-life of 18.3 s for binding at 4 degrees C. The saturable binding is specifically inhibited by tetrodotoxin with a K1 = 0.30 nM. The number of saturable binding sites in the extract is 97 fmol/mg protein. Since approx. 50% of the binding activity is recovered in the extract, the number of binding sites in the head is estimated to be 6.4 fmol/mg head. Nerve conduction in Drosophila larvae is completely blocked after 20 min in a bathing solution containing 200 nM tetrodotoxin. A comparison between the binding and the electrophysiological studies in Drosophila and other organisms suggests that the Drosophila saxitoxin receptor is part of the voltage-sensitive sodium channel involved in the propagation of action potentials. A mutant (ttxs), which is abnormally sensitive to dietary tetrodotoxin, is shown to be indistinguishable from wild type with respect to [3H]saxitonin-binding properties and physiological sensitivity to tetrodotoxin. These studies provide techniques which can be used to identify mutants with defects in the saxitoxin-binding component of the sodium channel.

Animals↗
PubMed1980

Effects of the dipolar form of phloretin on potassium conductance in squid giant axons.

The effects of phloretin on membrane ionic conductances have been studied in the giant axon of the squid, Loligo pealei. Phloretin reversibly suppresses the potassium and sodium conductances and modifies their dependence on membrane potential (Em). Its effects on the potassium conductance (GK) are much greater than on the sodium conductance; no effects on sodium inactivation are observed. Internal perfusion of phloretin produces both greater shifts in GK(Em) and greater reductions maximum GK than does external perfusion; the effect of simultaneous internal and external perfusion is little greater than that of internal perfusion alone. Lowering the internal pH, which favors the presence of the neutral species of weakly acidic phloretin (pKa 7.4), potentiates the actions of internally perfused phloretin. Other organic cations with dipole moments similar to phloretin's have little effect on either potassium or sodium conductances in squid axons. These results can be explained by either of two mechanisms; on postulates a phloretin "receptor" near the voltage sensor component of the potassium channel which is accessible to drug molecules applied at either the outer or inner membrane surface and is much more sensitive to the neutral than the negatively charged form of the drug. The other mechanism proposes that neutral phloretin molecules are dispersed in an ordered array in the membrane interior, producing a diffuse dipole field which modifies potassium channel gating. Different experimental results support these two mechanisms, and neither hypothesis can be disproven.

Animals↗
PubMed1980

Saxitoxin binding to sodium channels of rat skeletal muscles.

1. The saturable binding of exchange-labelled tritiated saxitoxin (STX) to the extensor digitorum longus (e.d.l.), diaphragm and soleus muscles of adult rats was studied. By measuring STX uptake to small pieces of muscle, the dissociation constant (KD) and binding capacity could be determined for individual muscles. 2. The affinity for STX is very similar in all three muscles, with a KD of 4.3 +/- 0.3, 5.1 +/- 0.5 and 4.9 +/- 0.5 nM (mean +/- S.E. of mean) at 4 degrees C for e.d.l., diaphragm and soleus respectively. The maximum binding capacity, which varies between different muscles, is 52.6 +/- 2.5, 40.3 +/- 4.9 and 23.8 +/- 1.1 f-mole.mg wet wt.-1 respectively. 3. The affinity of e.d.l. for tetrodotoxin (TTX), measured by inhibition of STX binding, is 12.1 +/- 1.4 nM at 4 degrees C. Raising the temperature to 37 degrees C increases the KD for STX to 6.8 +/- 0.8 nM and the KD for TTX to 47.5 +/- 4.5 nM. 4. STX binding is pH dependent; protons compete with STX for the binding site as if there were a titratable acidic group with a pK of 5.5. 5. The binding capacity of the diaphragm is not uniform along the length of the muscle fibres. Binding at the ends of the fibres is only 78% of that in the central region. 6. Denervation of e.d.l. for 7 days causes no change in the affinity for STX. There is a slight reduction in the binding capacity from 54 +/- 5 to 43 +/- 3 f-mole.mg wet wt.-1. There is no change in the diameter of the muscle fibres.

Animals↗
PubMed1979

Saxitoxin binding to synaptosomes, membranes, and solubilized binding sites from rat brain.

Binding of 3H-saxitoxin to Na+ channels was studied in subcellular fractions prepared from rat brain homogenates. Saxitoxin binding to synaptosomes was saturable with an apparent dissociation constant of about 1 nM; about 1 pmol/mg protein was bound at saturating saxitoxin concentrations. A linear, nonsaturable component of saxitoxin binding accounted for less than 3% of the total binding at 30 nM. Saxitoxin binding to synaptosomes was unaffected by depolarization with elevated K+ concentrations, or by activation of the Na+ channels with batrachotoxin plus a purified polypeptide toxin from the scorpion Leiurus quinquestriatus. A procedure is described for preparing a membrane fraction that contains 70--80% of the total saxitoxin binding activity of the crude homogenate. The specific activity of this fraction was about 4 to 6 pmol/mg protein. About 60--70% of the saxitoxin binding sites were solubilized by incubating these membranes with the nonionic detergent Triton X-100; the detergent-solubilized binding sites eluted at a position corresponding to a mol wt of about 700,000 on gel filtration chromatography. Both membrane-bound and solubilized saxitoxin binding were assayed by a new cation exchange column method. The binding of saxitoxin to both membrane-bound and detergent-solubilized binding sites was saturable with an apparent dissociation constant of about 2 nM. Dissociation of the saxitoxin-receptor complex followed a single exponential decay with a rate constant at 0 degrees of 0.1 min-1 for membrane bound and 0.2 min-1 for detergent-solubilized binding sites. The measured association rate constant was 6 X 10(8) M-1 min-1 at 0 degrees for membrane-bound saxitoxin binding sites.

Animals↗
PubMed1979

Binding of radioactively labeled saxitoxin to the squid giant axon.

The binding of saxitoxin, a specific inhibitor of the sodium conductance in excitable membranes, has been measured in giant axons from the squid, Loligo pealei. Binding was studied by labeling saxitoxin with tritium, using a solvent-exchange technique, and measuring the toxin uptake by liquid scintillation counting. Total toxin binding is the sum of a saturable, hyperbolic binding component, with a dissociation constant at 2--4 degrees C of 4.3 +/- 1.7 nM (mean SE), and a linear, nonsaturable component. The density of saturable binding sites is 166 +/- 20.4 micrometers-2. From this density and published values of the maximum sodium conductance, the conductance per toxin site is estimated to be about 7 pS, assuming sequential activation and inactivation processes (F. Bezanilla & C.M. Armstrong, 1977, J. Gen. Physiol. 70:549). This single site conductance value of 7 pS is in close aggreement with estimates of the conductance of one open sodium channel from measurements of gating currents and of noise on squid giant axons and is consistent with the hypothesis that one saxitoxin molecule binds to one sodium channel.

Animals↗
PubMed1979

Sodium channels in axons and glial cells of the optic nerve of Necturus maculosa.

Experiments investigating both the binding of radioactively labelled saxitoxin (STX) and the electrophysiological response to drugs that increase the sodium permeability of excitable membranes were conducted in an effort to detect sodium channels in glial cells of the optic nerve of Necturus maculosa, the mudpuppy. Glial cells in nerves from chronically enucleated animals, which lack optic nerve axons, show no saturable uptake of STX whereas a saturable uptake is clearly present in normal optic nerves. The normal nerve is depolarized by aconitine, batrachotoxin, and veratridine (10(-6)-10(-5) M), whereas the all-glial preparation is only depolarized by veratridine and at concentrations greater than 10(-3) M. Unlike the depolarization caused by veratridine in normal nerves, the response in the all-glial tissue is not blocked by tetrodotoxin nor enhanced by scorpion venom (Leiurus quinquestriatus). In glial cells of the normal nerve, where axons are also present, the addition of 10(-5) M veratridine does lead to a transient depolarization; however, it is much briefer than the axonal response to veratridine in this same tissue. This glial response to veratridine could be caused by the efflux of K+ from the drug-depolarized axons, and is similar to the glial response to extracellular K+ accumulation resulting from action potentials in the axon.

Animals↗
PubMed1977

On the voltage-dependent action of tetrodotoxin.

The use of the maximum rate-of-rise of the action potential (Vmax) as a measure of the sodium conductance in excitable membranes is invalid. In the case of membrane action potentials, Vmax depends on the total ionic current across the membrane; drugs or conditions that alter the potassium or leak conductances will also affect Vmax. Likewise, long-term depolarization of the membrane lessens the fraction of total ionic current that passes through the sodium channels by increasing potassium conductance and inactivating the sodium conductance, and thereby reduces the effect of Vmax of drugs that specifically block sodium channels. The resultant artifact, an apparent voltage-dependent potency of such drugs, is theoretically simulated for the effects of tetrodotoxin on the Hodgkin-Huxley squid axon.

Action Potentials↗
PubMed1976

A new method for labelling saxitoxin and its binding to non-myelinated fibres of the rabbit vagus, lobster walking leg, and garfish olfactory nerves.

1. A new method of labelling saxitoxin (STX) is described, based on transfer of tritium from tritiated water to the toxin. 2. The radiochemical purity of the labelled toxin has been directly determined, rather than being based on indirect biochemical means, as in previous experiments with Wilzbach-labelled STX and TTX. 3. The specific activity of the labelled toxin, 66 d..m.f-mole-1, corresponds with one tritium atom per molecule STX, an improvement of about 300-fold over other means of labelling TTX and STX. 4. The binding of this toxin to rabbit, lobster and garfish olfactory nerve fibres has been re-examined. 5. The density of sodium channels calculated on the basis of the binding of the toxin is about four to six-times the values previously reported.

Animals↗
PubMed1974

Evidence that tetrodotoxin and saxitoxin act at a metal cation binding site in the sodium channels of nerve membrane.

The effects of monovalent, divalent, and trivalent cations on the binding of tetrodotoxin and saxitoxin to intact nerves and to a preparation of solubilized nerve membranes have been examined. All eight divalent and trivalent cations tested, and the monovalent ions Li(+), Tl(+), and H(+) appear to compete reversibly with the toxins for their binding site. The ability of lithium to reduce toxin binding is paralleled by its ability to reduce tetrodotoxin-sensitive ion fluxes through the nerve membrane. We conclude that the toxins act at a metal cation binding site in the sodium channel and suggest that this site is the principal coordination site for cations (normally Na(+) ions) as they pass through the membrane during an action potential. The dissociation constant for Li(+) is 0.1-0.2 M and for Na(+) > 0.6 M, reflecting the weak binding necessary for rapid passage of sodium ions through the channel.

Animals↗
PubMed1973

The inhibition of sodium currents in myelinated nerve by quaternary derivatives of lidocaine.

The inhibition of sodium currents by quaternary derivatives of lidocaine was studied in single myelinated nerve fibers. Membrane currents were diminished little by external quaternary lidocaine (QX). QX present in the axoplasm (<0.5 mM) inhibited sodium currents by more than 90%. Inhibition occurred as the sum of a constant, tonic phase and a variable, voltage-sensitive phase. The voltage-sensitive inhibition was favored by the application of membrane potential patterns which produce large depolarizations when sodium channels are open. Voltage-sensitive inhibition could be reversed by small depolarizations which opened sodium channels. One explanation of this observation is that QX molecules enter open sodium channels from the axoplasmic side and bind within the channels. The voltage dependence of the inhibition by QX suggests that the drug binds at a site which is about halfway down the electrical gradient from inside to outside of the sodium channel.

Animals↗
PubMed1973

The binding of labelled saxitoxin to the sodium channels in nerve membranes.

1. Tritium labelled saxitoxin has been prepared and purified, and its binding both to intact rabbit vagus nerves and to a solubilized preparation of garfish olfactory nerve membranes has been examined.2. In intact and solubilized nerves there is a saturable binding component of magnitude equal to that previously obtained for labelled tetrodotoxin.3. This component of bound saxitoxin is displaced competitively by tetrodotoxin, and it is concluded that the two toxins bind to the same site.4. The saturable saxitoxin (STX) interaction with the nerve membrane is reversible and can be described by the equation STX + R right harpoon over left harpoon STX.R where R is the binding site or receptor. With the solubilized preparation of garfish nerve membranes the saxitoxin-receptor reaction rates are almost four times faster than those of tetrodotoxin. The half-life of the saxitoxin-receptor complexes is 13 sec compared with 44 sec for the tetrodotoxin-receptor complex.5. A number of agents were tested for their ability to displace the labelled saxitoxin. Calcium and thallous ions each produced significant reversible reduction in binding, with apparent equilibrium dissociation constants of about 20-30 mM. Toxin binding is also inhibited reversibly in acidic solutions by protons competing with toxin for a binding site with a pK(a) of 5.6-5.9. All three ions are known to block sodium currents in myelinated nerve at similar concentrations. Our experiments indicate that they do so at the site of toxin binding.6. Lidocaine and veratrine do not affect the binding of saxitoxin.

Animals↗
PubMed1971

The Necessity for beta-Carotene in the 518 Nanometer Absorbance Change.

The identity of the pigment responsible for the light-induced 518 nanometer absorbance change was investigated by extraction and reconstitution of spinach chloroplasts. Heptane extraction of carotene and quinones from lyophilized chloroplasts removes absorbance changes at 518 and 475 nanometers activated by both laser flash and continuous illumination. Some electron transport activity is always present, even in carotene- and quinone-depleted chloroplasts, but the light-induced pH increase disappears following the first extraction step. Readdition of pure beta-carotene partially restores the 518 and 475 nanometer absorbance changes.

Journal Article↗
PubMedDate not supplied

The local anesthetic properties and toxicity of saxitonin homologues for rat sciatic nerve block in vivo.

BACKGROUND AND OBJECTIVES: Saxitoxin and its homologues are naturally occurring compounds that block the sodium channel with high potency. They have the potential for providing prolonged duration local anesthesia when coinjected with vasoconstrictors or conventional local anesthetics and are devoid of local neurotoxicity. Here, we compare sciatic nerve block with saxitoxin to those with neosaxitoxin, decarbamoyl saxitoxin, and tetrodotoxin (TTX), in a search for even safer compounds. METHODS: Rats received percutaneous sciatic nerve block with toxins. The compounds were compared in terms of lethality, onset and duration of action for thermal analgesia (hot-plate testing), and motor block (weight-bearing). Data were expressed as medians with 25th and 75th percentiles, and median effective concentrations were determined. RESULTS: The median concentrations at which analgesia of 60 minutes duration was achieved were neosaxitoxin, 34+/-2 micromol/L; saxitoxin, 58+/-3 micromol/L; TTX, 92+/-5 micromol/L; and decarbamoyl saxitoxin, 268+/-8 micromol/L. Similar trends were observed for other measures of effectiveness (block duration of 90 minutes, maximal block), and for lethality so that the therapeutic indices were similar. No toxin had a marked predominance of sensory or motor block. The potency of TTX was intermediate between those of the saxitoxins, and its therapeutic index was slightly better. No difference was observed in time to onset of nerve blockade among the toxins. CONCLUSIONS: Substitutions on the saxitoxin nucleus result in large differences in incidence and duration of block, and toxicity. The therapeutic indices of the saxitoxins are similar; that of TTX is slightly better.

Anesthetics, Local↗