Predictions of the EC50 for action potential block for aliphatic solutes.

Experiments were conducted to test the hypothesis that aliphatic hydrocarbons bind to pockets/crevices of sodium (Na(+)) channels to cause action potential (AP) block. Aliphatic solutes exhibiting successively greater octanol/water partitition coefficients (K (ow)) were studied. Each solute blocked Na(+) channels. The 50% effective concentration (EC(50)) to block APs could be mathematically predicted as a function of the solute's properties. The solutes studied were methyl ethyl ketone (MEK), cyclohexanone, dichloromethane, chloroform and triethylamine (TriEA); the K (ow) increased from MEK to TriEA. APs were recorded from frog nerves, and test solutes were added to Ringer's solution bathing the nerve. When combined with EC(50)s for solutes with log K (ow)s < 0.29 obtained previously, the solute EC(50)s could be predicted as a function of the fractional molar volume (dV/dm = [dV/dn]/100), polarity (P) and the hydrogen bond acceptor basicity (beta) by the following equation: EC(50) = 2.612({-2.117[dv/dm]+0.6424P+2.628 beta}) Fluidity changes cannot explain the EC(50)s. Each of the solutes blocks Na(+) channels with little or no change in kinetics. Na(+) channel block explains much of the EC(50) data. EC(50)s are produced by a combination of effects including ion channel block, fluidity changes and osmotically induced structural changes. As the solute log K (ow) increases to values near 1 or greater, Na(+) channel block dominates in determining the EC(50). The results are consistent with the hypothesis that the solutes bind to channel crevices to cause Na(+) channel and AP block.

Isolation, purification and N-terminal sequencing of a bioactive peptide that alters action potentials from the venom of Buthus martensii Karsch.

A bioactive peptide that extensively prolongs action potentials (APs) in frog nerve has been isolated and purified from the venom of the scorpion Buthus martensii Karsch (BMK). The peptide, designated as BMK 18(2), was purified using gel filtration, ion exchange, FPLC, and HPLC chromatography. APs recorded in the presence of nanomolar concentrations of the peptide were extensively prolonged with some attenuation in their heights. The N-terminal sequence of BMK 18(2) was found to be: VRDAYIAEDYD-VYH-ARDA. Sequence similarity comparisons to other alpha-scorpion toxins suggest that the two blanks in the sequences are cysteines. The molecular weight (M.W.) of BMK 18(2) was determined by LC/MS/MS to be 7185 Da. Since the peptide prolongs APs when both K+ and Ca++ channels were blocked and shows sequence similarity to other alpha-neurotoxins, it appears likely that BMK 18(2) acts to alter Na channel inactivation to produce its effect.

ED50 G(Na) block predictions for phenyl substituted and unsubstituted n-alkanols.

To study the role the phenyl group plays in producing local anesthetic block, a sequence of n-alkanols and phenyl-substituted alkanols (phi)-alkanols) were characterized in their ability to block Na channels. The sequence of n-alkanols studied possess 3-5 carbons (propanol-pentanol). The action of phenol and 3-phi-alkanols (benzyl alcohol, phenethyl alcohol, 3-phenyl-1-propanol) were also studied. Na currents (INa) were recorded from single frog skeletal muscle fibers using the Vaseline-gap voltage clamp technique. INas were recorded prior to, during, and following the removal of the solutes in Ringer's solution. All alkanols and phenol acted to block INa in a dose-dependent manner. Effective doses to produce half block (ED50) of INa or Na conductance (GNa) were obtained from dose-response relations for all solutes used. The block of GNa depended on voltage, and could be separated into voltage-dependent and -independent components. Each solute acted to shift GNa-V relations in a depolarized direction and reduce the maximum GNa and slope of the relation. All solutes acted to speed up INa kinetics and cause hyperpolarizing shifts in steady-state inactivation. The magnitude of the kinetic changes increased with dose. Size was an important variable in determining the magnitude of the changes in INa; however, size alone was not sufficient to predict the changes in INa. ED50s for GNa and AP block could be predicted as a function of intrinsic molar volume, hydrogen bond acceptor basicity (beta) and donor acidity (alpha), and polarity (P) of the solutes. The equivalency of ED50 predictions for AP and GNa block can be explained by the fact that AP block arises from channel block and solute-induced changes in INa kinetics. phi-Alkanols were more effective at blocking and inactivating Na channels than their unsubstituted counterparts. Phenyl-substituted alkanols are more likely to interact with the channel than their unsubstituted counterparts.

ED50 AP block predictions for phenyl substituted and unsubstituted n-alkanols.

A series of n-alkanols and phenyl-substituted n-alkanols (phi-alkanols) of increasing chain length and phenol were characterized for their ability to block action potentials (APs) in frog sciatic nerves. APs were recorded using the single sucrose-gap method. The degree of AP attenuation when the nerve was exposed to different concentrations of an alcohol was used to construct dose-response curves. The reciprocals of the half-blocking doses (ED50s) were used to obtain a measure of the potency of the alcohols. For n-alkanols and phi-alkanols, increasing the chain length by the addition of a methylene group increased the potency on average by 3.1 for both groups of alkanols. The addition of a phenyl group caused a potency increase that ranged between the values of 77 and 122. The ED50 for both groups of alkanols could not be solely predicted by the log octanol-water partition coefficient (Kow). Using linear solvation energy relations (LSER), the log ED50 could be described as a linear combination of the intrinsic (van der Waals) molar volume (VI), polarity (P), and hydrogen bond acceptor basicity (beta) and donor acidity (alpha). Size alone could not predict the ED50 for both n-alkanols and phi-alkanols. The results are consistent with the hypothesis that alkanols bind to and interact with Na channels to cause AP block. Phenyl group addition to an alkanol markedly increases the molecule's potency.

The isolation and purification of two peptides from the venom of Buthus martensii Karsch.

Two peptides that extensively prolong action potentials (APs) in rat and frog nerves have been isolated and purified from the venom of the scorpion Buthus martensii Karsch (BMK). The peptides were purified using gel filtration, ion exchange, FPLC, and HPLC chromatography. Action potentials recorded in the presence of nanomolar concentrations of the peptides were extensively prolonged without much attenuation in their heights. The N-terminal sequences of both the peptides, BMK 9(3)-1 and BMK 9(3)-2, were determined. The N-terminal sequences of BMK 9(3)-1 and BMK 9(3)-2 were found to be: GRDAYIADSEN-PYF-GANPN and GRDAYIADSEN-PYT-ALNP. Sequence similarity comparisons to other alpha-scorpion toxins suggest that the two blanks in each of the sequences are cysteines. The first 20 residues of the two BMK peptides differ by only three amino acid substitutions. The molecular weight (MW) of BMK 9(3)-1 and BMK 9(3)-2 were determined by LC/MS/MS to be 7020 and 7037 Da. Since both of the peptides prolong APs when both K(+) and Ca(++) channels are blocked and show sequence similarity to other alpha-neurotoxins, it appears likely that BMK 9(3)-1 and BMK 9(3)-2 act to alter Na channel inactivation to produce their effects. The first 20 residues of BMK 9(3)-2 are identical to those observed for makatoxin I, a toxin isolated from Buthus martensii Karsch venom, that alters nitric oxide transmitter release. Since the two toxins also have very similar molecular weights, BMK 9(3)-2 may be identical to makatoxin I; however, BMK 9(3)-2 acts to alter Na channels to exert its effect, thus the two toxins may differ, or if they are identical, they can exert effects on both neural transmission and AP propagation.

The isolation and characterization of a peptide that alters sodium channels from Buthus martensii Karsch.

The peptides were purified using gel filtration, ion exchange, FPLC, and HPLC chromatography and found to greatly prolong action potentials at nanomolar concentrations when applied to frog and mouse nerves. The N-terminal primary amino acid sequence of one of the peptides, BMK 16(5), was determined. The first 23 amino acids of BMK 16(5) were found to be: VKDGYIADDRNCPYFCGRNAYYD. The two cysteine residues in the sequence appeared as Edman sequence cycle blanks; however, they were assigned to be cysteines due to sequence similarity to other peptide toxins that bind to sodium channels and identification of the presence of cysteines obtained from single time point amino acid analysis. The MW of BMK 16(5) was determined by a Perkin Elmer API 300 LC/MS/MS to be 3,695. The amino acid residues of BMK 16(5) show strong similarity with the first 23 amino acid residues of a number of scorpion alpha neurotoxins. Unlike these neurotoxins, BMK 16(5) possesses a proline residue at position 13 which will likely make it fold in a unique way so as to bind to and alter sodium channels.

An analysis of dimethylsulfoxide-induced action potential block: a comparative study of DMSO and other aliphatic water soluble solutes.

A series of water soluble aliphatic solutes were chosen for study. Fifty percent effective doses (ED50) to block propagated compound action potentials (AP's) were obtained by examining dose-response relations for each solute. All solutes used were liquids at room temperature and are typically used as solvents. The solutes studied were dimethylsulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide, acetone, and hexamethylphosphoramide (HMPA); the octanol/water partition coefficients for these test substances form an ordered sequence that increased 40-fold from DMSO to HMPA. AP's were recorded from desheathed frog sciatic nerves using the sucrose-gap technique; test solutes were added to Ringer's solution and applied externally to the nerve. ED50's for the solutes could be predicted as a function of the molar volume (dV/dn), polarity (P), and the hydrogen bond acceptor basicity (beta). Voltage-clamp experiments employing the vaseline-gap technique on single muscle fibers showed that each solute reduced Na+ current with little change in their kinetics at all voltages studied. Experiments using DMSO or DMF showed that Na+ channel block alone is insufficient to explain the respective ED50 values of AP block. Experiments conducted using a chloride transport-sensitive membrane fluidity assay, using rat pancreas secretory granules, suggested that each of the solutes act to increase membrane fluidity at doses below and above ED50 values. Light microscopic observations of fixed thick sections of whole nerves previously exposed to DMSO or DMF show structural changes; however, ED50 values cannot be simply explained by osmotic alterations of nerve structure. ED50's are likely to be produced by a combination of effects including osmotically induced nerve structural changes, ion channel block, and fluidity changes. The toxicity (lethal doses or toxic concentrations) of each of these five solutes correlates well with the ED50 and could be predicted as a function of dV/dn, P, and beta.

Purification of protein toxins from Leiurus quinquestriatus hebraeus that modify Na channels.

Two protein toxins (Lqh1 and Lqh2) were purified from crude venom obtained from Middle Eastern scorpions, Leiurus quinquestriatus hebraeus, by using cationic exchange chromatography. Lqh1 and Lqh2 were compared to toxin V (Lqq5) obtained from the venom of the North African scorpion Leiurus quinquestriatus quinquestriatus. Lqh1 and Lqh2 were purified to homogeneity; they had mol. wts of 6390 and 5870, respectively; thus both toxins differ in size from Lqq5 (7462). Electrophysiological experiments also suggested that all three toxins are different. In a dose-dependent manner, Lqh1, Lqh2 and Lqq5 lengthened and attenuated propagated compound action potentials (AP) recorded from frog sciatic nerves using the single sucrose-gap technique. Toxins Lqh1 and Lqh2 were found to be more effective than Lqq5 in both lengthening and blocking APs. Voltage-clamp experiments using the vaseline-gap technique on frog skeletal muscle fibres showed that Lqh1 and Lqh2 attenuated the Na current amplitude and slowed inactivation, while Lqq5 primarily lengthened the Na current duration. Increases in the holding potential increase the current attenuation caused by all three toxins. Evidence from sucrose-gap and voltage-clamp experiments suggests that all three toxins bind to Na channels and block them, besides their well-known ability to slow inactivation kinetics. The increased effectiveness of Lqh1 appears to be produced by a slowed rate of exit of the toxin from its binding site.

Alterations in sodium channel gating produced by the venom of the marine mollusc Conus striatus.

The action of the venom from the marine mollusc Conus striatus was studied using the voltage-clamp technique on myelinated nerve. Conus venom applied to an isolated node of Ranvier at 1.1 micrograms protein/ml produced repetitive firing of action potentials when the node was depolarized under current-clamp conditions. Venom application produced a leftword (depolarizing) shift in both the peak sodium current-voltage and the permeability-voltage relationships. A concomitant decrease in maximum peak current and permeability also occurred. The time course of sodium current decline (inactivation) was slowed at all voltages by the presence of venom. Venom treatment caused only a slight depolarizing shift (5 mV) in the voltage-dependence of steady-state Na inactivation. The closing to the resting state of previously activated Na channels, "deactivation", was judged from Na "tail" currents following membrane repolarization, and was slowed more than four-fold by venom treatment. The changes in Na channel gating produced by Conus striatus venom can best be described as a stabilization of the open state of the Na channel and a shift in the voltage dependence of the opening of Na channels. The slowing of both inactivation and deactivation of Na channels can be simulated by alterations in the rate constants of a five state Markov model.

Na activation delays and their relation to inactivation in frog skeletal muscle.

Delays in the development of activation of Na currents were studied using voltage-clamped frog skeletal muscle fibers. Na currents elicited by a depolarizing voltage step from a hyperpolarized membrane potential were delayed in their activation when compared to Na currents elicited from the resting potential. The magnitude of the delay increased with larger hyperpolarizing potentials and decreased with larger depolarizing test potentials. Delays in activation observed following chloramine-T treatment that partially removes inactivation did not differ from delays observed before treatment. Longer exposures of the muscle fiber to chloramine-T led to a complete loss of inactivation, coincident with an elimination of the hyperpolarization-induced delays in activation. Steady-state slow inactivation was virtually unaffected by prolonged exposures of the fibers to chloramine-T that eliminated fast inactivation. The results show that chloramine-T acts at a number of sites to alter both activation and inactivation. Markov model simulations of the results show that chloramine-T alters fundamental time constants of the system by altering both activation and inactivation rate constants.

Removal of inactivation causes time-invariant sodium current decays.

The kinetic properties of the closing of Na channels were studied in frog skeletal muscle to obtain information about the dependence of channel closing on the past history of the channel. Channel closing was studied in normal and modified channels. Chloramine-T was used to modify the channels so that inactivation was virtually removed. A series of depolarizing prepulse potentials was used to activate Na channels, and a -140-mV postpulse was used to monitor the closing of the channels. Unmodified channels decay via a biexponential process with time constants of 72 and 534 microseconds at 12 degrees C. The observed time constants do not depend upon the potential used to activate the channels. The contribution of the slow component to the total decay increases as the activating prepulse is lengthened. After inactivation is removed, the biexponential character of the decay is retained, with no change in the magnitude of the time constants. However, increases in the duration of the activating prepulse over the range where the current is maximal 1-75 ms) produce identical biexponential decays. The presence of biexponential decays suggests that either two subtypes of Na channels are found in muscle, or Na channels can exist in one of two equally conductive states. The time-invariant decays observed suggest that channel closure does not depend upon their past history.

Altered sodium and gating current kinetics in frog skeletal muscle caused by low external pH.

The effect of low pH on the kinetics of Na channel ionic and gating currents was studied in frog skeletal muscle fibers. Lowering external pH from 7.4 to 5.0 slows the time course of Na current consistent with about a +25-mV shift in the voltage dependence of activation and inactivation time constants. Similar shifts in voltage dependence adequately describe the effects of low pH on the tail current time constant (+23.3 mV) and the gating charge vs. voltage relationship (+22.1 mV). A significantly smaller shift of +13.3 mV described the effect of pH 5.0 solution on the voltage dependence of steady state inactivation. Changes in the time course of gating current at low pH were complex and could not be described as a shift in voltage dependence. tau g, the time constant that describes the time course of the major component of gating charge movement, was slowed in pH 5.0 solution by a factor of approximately 3.5 for potentials from -60 to +45 mV. We conclude that the effects of low pH on Na channel gating cannot be attributed simply to a change in surface potential. Therefore, although it may be appropriate to describe the effect of low pH on some Na channel kinetic properties as a "shift" in voltage dependence, it is not appropriate to interpret such shifts as a measure of changes in surface potential. The maximum gating charge elicited from a holding potential of -150 mV was little affected by low pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Simple shifts in the voltage dependence of sodium channel gating caused by divalent cations.

The effect of elevated divalent cation concentration on the kinetics of sodium ionic and gating currents was studied in voltage-clamped frog skeletal muscle fibers. Raising the Ca concentration from 2 to 40 mM resulted in nearly identical 30-mV shifts in the time courses of activation, inactivation, tail current decay, and ON and OFF gating currents, and in the steady state levels of inactivation, charge immobilization, and charge vs. voltage. Adding 38 mM Mg to the 2 mM Ca bathing a fiber produced a smaller shift of approximately 20 mV in gating current kinetics and the charge vs. voltage relationship. The results with both Ca and Mg are consistent with the hypothesis that elevated concentrations of these alkali earth cations alter Na channel gating by changing the membrane surface potential. The different shifts produced by Ca and Mg are consistent with the hypothesis that the two ions bind to fixed membrane surface charges with different affinities, in addition to possible screening.

Effects of deuterium oxide on the rate and dissociation constants for saxitoxin and tetrodotoxin action. Voltage-clamp studies on frog myelinated nerve.

The actions of tetrodotoxin (TTX) and saxitoxin (STX) in normal water and in deuterium oxide (D2O) have been studied in frog myelinated nerve. Substitution of D2O for H2O in normal Ringer's solution has no effect on the potency of TTX in blocking action potentials but increases the potency of STX by approximately 50%. Under voltage clamp, the steady-state inhibition of sodium currents by 1 nM STX is doubled in D2O as a result of a halving of the rate of dissociation of STX from the sodium channel; the rate of block by STX is not measurably changed by D2O. Neither steady-state inhibition nor the on- or off-rate constants of TTX are changed by D2O substitution. The isotopic effects on STX binding are observed less than 10 min after the toxin has been added to D2O, thus eliminating the possibility that slow-exchange (t 1/2 greater than 10 h) hydrogen-binding sites on STX are involved. The results are consistent with a hypothesis that attributes receptor-toxin stabilization to isotopic changes of hydrogen bonding; this interpretation suggests that hydrogen bonds contribute more to the binding of STX than to that of TTX at the sodium channel.

Initial conditions and the kinetics of the sodium conductance in Myxicola giant axons. I. effects on the time-course of the sodium conductance.

The effects of conditioning polarizations, ranging from--150 to 0 mV and of durations from 50 mus to 30 ms, on the time-course of GNa during test steps in potential were studied in Myxicola giant axons. Beyond the effects of conditioning polarizations on the amplitude of GNa, the only effect was to produce a translation of GNa(t) along the time axis without a change in shape. For depolarizing conditioning potentials, Hodgkin-Huxley kinetics predict time shifts about threefold greater than found experimentally, whereas the predictions of the coupled model of Goldman (1975. Biophys. J. 15:119--136) were in approximate agreement with our experiments. The time shifts developed over an exponential time-course as the conditioning pulse duration was increased. The time constant of development of the time shift was considerably faster than, and showed the opposite dependency on potential from, the values predicted by both models. It had a mean Q10 of 1/2.50. This fast activation process cannot account for the observed rise time behavior of GNa, suggesting that there is an additional activation process. All results are consistent with the idea that the gating structure displays more than three states, with state intermediate between rest and conducting.

Initial conditions and the kinetics of the sodium conductance in Myxicola giant axons. II. Relaxation experiments.

The time-course of the decay of INa on resetting the membrane potential to various levels after test steps in potential was studied. The effects of different initial conditions on these Na tail currents were also studied. For postpulse potentials at or negative to -35 mV, these currents may be attributed nearly entirely to the shutdown of the activation process, inactivation being little involved. Several relaxations may be detected in the tail currents. The slower two are well defined exponentials with time constants of approximately 1 ms and 100 mus in the hyperpolarizing potential range. The fastest relaxation is only poorly resolved. Different initial conditions could alter the relative weighting factors on the various exponential terms, but did not affect any of the individual time constants. The activation of the sodium conductance cannot be attributed to any number of independent and identical two-state subunits with first order transitions. The results of this and the previous paper are discussed in terms of the minimum kinetic scheme consistent with the data. Evidence is also presented suggesting that there may exist a small subpopulation of channels with different kinetics and a faster rate of recovery from TTX block than the rest of the population.