THE PRODUCTION AND INHIBITION OF ACTION CURRENTS BY ALCOHOL

Suitable concentrations of ethyl alcohol (1 to 1.5 M) applied to a spot on a cell of Nitella lower the P.D. enough to cause action currents. The alcohol then suppresses action currents arriving from other parts of the cell and acts as a block. After the alcohol is removed the normal P.D. and irritability return. Similar experiments on the sciatic nerve and skin of the frog produced only a negative result.     

RESTORATION OF THE POTASSIUM EFFECT BY MEANS OF ACTION CURRENTS

Treatment with distilled water removes from Nitella the ability to give the large potential difference between 0.01 M KCl and 0.01 M NaCl which is known as the potassium effect. The potassium effect may be restored by action currents. This might be explained by saying that distilled water removes from the surface a substance, R, which is responsible for the potassium effect and which moves into the surface during the action current and thereby restores the potassium effect.     

THE EFFECTS OF CURRENT FLOW ON BIOELECTRIC POTENTIAL : III. NITELLA

String galvanometer records show the effect of current flow upon the bioelectric potential of Nitella cells. Three classes of effects are distinguished. 1. Counter E.M.F''S, due either to static or polarization capacity, probably the latter. These account for the high effective resistance of the cells. They record as symmetrical charge and discharge curves, which are similar for currents passing inward or outward across the protoplasm, and increase in magnitude with increasing current density. The normal positive bioelectric potential may be increased by inward currents some 100 or 200 mv., or to a total of 300 to 400 mv. The regular decrease with outward current flow is much less (40 to 50 mv.) since larger outward currents produce the next characteristic effect. 2. Stimulation. This occurs with outward currents of a density which varies somewhat from cell to cell, but is often between 1 and 2 µa/cm.² of cell surface. At this threshold a regular counter E.M.F. starts to develop but passes over with an inflection into a rapid decrease or even disappearance of positive P.D., in a sigmoid curve with a cusp near its apex. If the current is stopped early in the curve regular depolarization occurs, but if continued a little longer beyond the first inflection, stimulation goes on to completion even though the current is then stopped. This is the "action current" or negative variation which is self propagated down the cell. During the most profound depression of P.D. in stimulation, current flow produces little or no counter E.M.F., the resistance of the cell being purely ohmic and very low. Then as the P.D. begins to recover, after a second or two, counter E.M.F. also reappears, both becoming nearly normal in 10 or 15 seconds. The threshold for further stimulation remains enhanced for some time, successively larger current densities being needed to stimulate after each action current. The recovery process is also powerful enough to occur even though the original stimulating outward current continues to flow during the entire negative variation; recovery is slightly slower in this case however. Stimulation may be produced at the break of large inward currents, doubtless by discharge of the enhanced positive P.D. (polarization). 3. Restorative Effects.—The flow of inward current during a negative variation somewhat speeds up recovery. This effect is still more strikingly shown in cells exposed to KCl solutions, which may be regarded as causing "permanent stimulation" by inhibiting recovery from a negative variation. Small currents in either direction now produce no counter E.M.F., so that the effective resistance of the cells is very low. With inward currents at a threshold density of some 10 to 20 µa/cm.², however, there is a counter E.M.F. produced, which builds up in a sigmoid curve to some 100 to 200 mv. positive P.D. This usually shows a marked cusp and then fluctuates irregularly during current flow, falling off abruptly when the current is stopped. Further increases of current density produce this P.D. more rapidly, while decreased densities again cease to be effective below a certain threshold. The effects in Nitella are compared with those in Valonia and Halicystis, which display many of the same phenomena under proper conditions. It is suggested that the regular counter E.M.F.''S (polarizations) are due to the presence of an intact surface film or other structure offering differential hindrance to ionic passage. Small currents do not affect this structure, but it is possibly altered or destroyed by large outward currents, restored by large inward currents. Mechanisms which might accomplish the destruction and restoration are discussed. These include changes of acidity by differential migration of H ion (membrane "electrolysis"); movement of inorganic ions such as potassium; movement of organic ions, (such as Osterhout''s substance R), or the radicals (such as fatty acid) of the surface film itself. Although no decision can be yet made between these, much evidence indicates that inward currents increase acidity in some critical part of the protoplasm, while outward ones decrease acidity.     

Novel nicotinic action of the sulfoximine insecticide sulfoxaflor

The novel sulfoximine insecticide sulfoxaflor is as potent or more effective than the neonicotinoids for toxicity to green peach aphids (GPA, Myzus persicae). The action of sulfoxaflor was characterized at insect nicotinic acetylcholine receptors (nAChRs) using electrophysiological and radioligand binding techniques. When tested for agonist properties on Drosophila melanogaster D??2 nAChR subunit co-expressed in Xenopus laevis oocytes with the chicken ??2 subunit, sulfoxaflor elicited very high amplitude (efficacy) currents. Sulfoximine analogs of sulfoxaflor were also agonists on D??2/??2 nAChRs, but none produced maximal currents equivalent to sulfoxaflor nor were any as toxic to GPAs. Additionally, except for clothianidin, none of the neonicotinoids produced maximal currents as large as those produced by sulfoxaflor. These data suggest that the potent insecticidal activity of sulfoxaflor may be due to its very high efficacy at nAChRs. In contrast, sulfoxaflor displaced [³H]imidacloprid (IMI) from GPA nAChR membrane preparations with weak affinity compared to most of the neonicotinoids examined. The nature of the interaction of sulfoxaflor with nAChRs apparently differs from that of IMI and other neonicotinoids, and when coupled with other known characteristics (novel chemical structure, lack of cross-resistance, and metabolic stability), indicate that sulfoxaflor represents a significant new insecticide option for the control of sap-feeding insects.     

NATURE OF THE ACTION CURRENT IN NITELLA : III. SOME ADDITIONAL FEATURES

Several forms of the action curve are described which might be accounted for on the ground that the outer protoplasmic surface shows no rapid electrical change. This may be due to the fact that the longitudinal flow of the outgoing current of action is in the protoplasm instead of in the cellulose wall. Hence the action curve has a short period with a single peak which does not reach zero. On this basis we can estimate the P.D. across the inner and outer protoplasmic surfaces separately. These P.D.''s can vary independently. In many cases there are successive action currents with incomplete recovery (with an increase or decrease or no change of magnitude). Some of the records resemble those obtained with nerve (including bursts of action currents and after-positivity).     

CALCULATIONS OF BIOELECTRIC POTENTIALS : IV. SOME EFFECTS OF CALCIUM ON POTENTIALS IN NITELLA

In Nitella the substitution of KCl for NaCl changes the P.D. in a negative direction. In some cases this change is lessened by adding solid CaCl₂ to the solution of KCl. This may be due to lessening the partition coefficient of KCl or to decreasing the solubility of an organic substance which sensitizes the cell to the action of KCl. Little or no correlation exists between this effect of calcium and its ordinary antagonistic action in producing a balanced solution which preserves the life of the cell indefinitely. CaCl₂ is negative to NaCl but positive to KCl. The effects of mixtures of KCl, NaCl, and CaCl₂ are discussed. The concentration effect of a mixture of KCl + CaCl₂ shows certain peculiarities due to action currents: these resemble those found with pure KCl. These studies and others on Nitella, Valonia, and Halicystis indicate that mobilities and partition coefficients are variable and can be brought under experimental control.     

THE EFFECTS OF CURRENT FLOW ON BIOELECTRIC POTENTIAL : II. HALICYSTIS

The effect of direct current, of controlled direction and density, across the protoplasm of impaled cells of Halicystis, is described. Inward currents slightly increase the already positive P.D. (70 to 80 mv.) in a regular polarization curve, which depolarizes equally smoothly when the current is stopped. Outward currents of low density produce similar curves in the opposite direction, decreasing the positive P.D. by some 10 or 20 mv. with recovery on cessation of flow. Above a critical density of outward current, however, a new effect becomes superimposed; an abrupt reversal of the P.D. which now becomes 30 to 60 mv. negative. The reversal curve has a characteristic shape: the original polarization passes into a sigmoid reversal curve, with an abrupt cusp usually following reversal, and an irregular negative value remaining as long as the current flows. Further increases of outward current each produce a small initial cusp, but do not greatly increase the negative P.D. If the current is decreased, there occurs a threshold current density at which the positive P.D. is again recovered, although the outward current continues to flow. This current density (giving positivity) is characteristically less than that required to produce reversal originally, giving the process a hysteretic character. The recovery is more rapid the smaller the current, and takes only a few seconds in the absence of current flow, its course being in a smooth curve, usually without an inflection, thus differing from the S-shaped reversal curve. The reversal produced by outward current flow is compared with that produced by treatment with ammonia. Many formal resemblances suggest that the same mechanism may be involved. Current flow was therefore studied in conjunction with ammonia treatment. Ammonia concentrations below the threshold for reversal were found to lower the threshold for outward currents. Subthreshold ammonia concentrations, just too low to produce reversal alone, produced permanent reversal when assisted by a short flow of very small outward currents, the P.D. remaining reversed when the current was stopped. Further increases of outward current, when the P.D. had been already reversed by ammonia, produced only small further increases of negativity. This shows that the two treatments are of equivalent effect, and mutually assist in producing a given effect, but are not additive in the sense of being superimposable to produce a greater effect than either could produce by itself. Since ammonia increases the alkalinity of the sap, and presumably of the protoplasm, when it penetrates, it is possible that the reversal of P.D. by current flow is also due to change of pH. The evidence for increased alkalinity or acidity due to current flow across phase boundaries or membranes is discussed. While an attractive hypothesis, it meets difficulties in H. ovalis where such pH changes are both theoretically questionable and practically ineffective in reversing the P.D. It seems best at the present time to assign the reversal of P.D. to the alteration or destruction of one surface layer of the protoplasm, with reduction or loss of its potential, leaving that at the other surface still intact and manifesting its oppositely directed potential more or less completely. The location of these surfaces is only conjectural, but some evidence indicates that it is the outer surface which is so altered, and reconstructed on recovery of positive P.D. This agrees with the essentially all-or-none character of the reversal. The various treatments which cause reversal may act in quite different ways upon the surface.     

THE EFFECTS OF CURRENT FLOW ON BIOELECTRIC POTENTIAL : I. VALONIA

The effect of direct current flow upon the potential difference across the protoplasm of impaled Valonia cells was studied. Current density and direction were controlled in a bridge which balanced the ohmic resistances, leaving the changes (increase, decrease, or reversal) of the small, normally negative, bioelectric potential to be recorded continuously, before, during, and after current flow, with a string galvanometer connected into a vacuum tube detector circuit. Two chief states of response were distinguished: State A.—Regular polarization, which begins to build up the instant current starts to flow, the counter E.M.F. increasing most rapidly at that moment, then more and more slowly, and finally reaching a constant value within 1 second or less. The magnitude of counter E.M.F. is proportional to the current density with small currents flowing in either direction across the protoplasm, but falls off at higher density, giving a cusp with recession to lower values; this recession occurs with slightly lower currents outward than inward. Otherwise the curves are much the same for inward and outward currents, for different densities, for charge and discharge, and for successive current flows. There is a slight tendency for the bioelectric potential to become temporarily positive following these current flows. Records in the regular state (State A) show very little effect of increased series resistance on the time constant of counter E.M.F. This seems to indicate that a polarization rather than a static capacity is involved. State B.—Delayed and non-proportional polarization, in which there is no counter E.M.F. developed with small currents in either direction across the protoplasm, nor with very large outward currents. But with inward currents a threshold density is reached at which a counter E.M.F. rather suddenly develops, with a sigmoid curve rising to high positive values (200 mv. or more). There is sometimes a cusp, after which the P.D. remains strongly positive as long as the current flows. It falls off again to negative values on cessation of current flow, more rapidly after short flows, more slowly after longer ones. The curves of charge are usually quite different in shape from those of discharge. Successive current flows of threshold density in rapid succession produce quicker and quicker polarizations, the inflection of the curve often becoming smoothed away. After long interruptions, however, the sigmoid curve reappears. Larger inward currents produce relatively little additional positive P.D.; smaller ones on the other hand, if following soon after, have a greatly increased effectiveness, the threshold for polarization falling considerably. The effect dies away, however, with very small inward currents, even as they continue to flow. Over a medium range of densities, small increments or decrements of continuing inward current produce almost as regular polarizations as in State A. Temporary polarization occurs with outward currents following soon after the threshold inward currents, but the very flow of outward current tends to destroy this, and to decondition the protoplasm, again raising the threshold, for succeeding inward flows. State A is characteristic of a few freshly gathered cells and of most of those which have recovered from injuries of collecting, cleaning, and separating. It persists a short time after such cells are impaled, but usually changes over to State B for a considerable period thereafter. Eventually there is a reappearance of regular polarization; in the transition there is a marked tendency for positive P.D. to be produced after current flow, and during this the polarizations to outward currents may become much larger than those to inward currents. In this it resembles the effects of acidified sea water, and of certain phenolic compounds, e.g. p-cresol, which produce State A in cells previously in State B. Ammonia on the other hand counteracts these effects, producing delayed polarization to an exaggerated extent. Large polarizations persist when the cells are exposed to potassium-rich solutions, showing it is not the motion of potassium ions (e.g. from the sap) which accounts for the loss or restoration of polarization. It is suggested that inward currents restore a protoplasmic surface responsible for polarization by increasing acidity, while outward currents alter it by increasing alkalinity. Possibly this is by esterification or saponification respectively of a fatty film. For comparison, records of delayed polarization in silver-silver chloride electrodes are included.     

PACEMAKERS IN NITELLA : II. ARRHYTHMIA AND BLOCK

Many forms of irregular rhythm and of partial block occurring in the vertebrate heart can be duplicated in Nitella. In order to observe these phenomena the cells of Nitella are kept for 6 weeks or more in a nutrient solution. They are then exposed for 3 hours or less to 0.01 M NaCl, NaSCN, or guanidine chloride, which reduce the time required for the action current to about 1 second (the normal time is 15 to 30 seconds). A pacemaker is established at one end of the cell by placing it in contact with 0.01 M KCl. This produces action currents at the rate of about 1 a second. Apparently some parts of the cell are unable to follow this rapid pace and hence fall into irregular rhythms (arrhythmia) and fail to register all the impulses (partial block).     

Diethylcarbamazine Increases Activation of Voltage-Activated Potassium (SLO-1) Currents in Ascaris suum and Potentiates Effects of Emodepside

Diethylcarbamazine is a drug that is used for the treatment of filariasis in humans and animals; it also has effects on intestinal nematodes, but its mechanism of action remains unclear. Emodepside is a resistance-busting anthelmintic approved for treating intestinal parasitic nematodes in animals. The novel mode of action and resistance-breaking properties of emodepside has led to its use against intestinal nematodes of animals, and as a candidate drug for treating filarial parasites. We have previously demonstrated effects of emodepside on SLO-1 K⁺-like currents in Ascaris suum. Here, we demonstrate that diethylcarbamazine, which has been proposed to work through host mediated effects, has direct effects on a nematode parasite, Ascaris suum. It increases activation of SLO-1 K⁺ currents and potentiates effects of emodepside. Our results suggest consideration of the combination of emodepside and diethylcarbamazine for therapy, which is predicted to be synergistic. The mode of action of diethylcarbamazine may involve effects on parasite signaling pathways (including nitric oxide) as well as effects mediated by host inflammatory mediators.     

PROTOPLASMIC POTENTIALS IN HALICYSTIS : IV. VACUOLAR PERFUSION WITH ARTIFICIAL SAP AND SEA WATER

Perfusion of the vacuole of living cells of Halicystis is described, the method employing two longitudinally fused capillaries as entrance and exit tubes. Natural sap, artificial sap, and sea water have been successfully perfused, with various additions and deficiencies, within the limits of physiological balance. In H. ovalis the P.D. remains positive and scarcely reduced in value when normal sea water, at pH 8.1, is perfused in the vacuole. In H. Osterhoutii the P.D. reverses in sign when the perfused solution has a higher pH than 6.5. In both cases a large P.D. persists when the solutions are the same on both sides of the protoplasm. In the absence of external gradients, there must be some internal gradient or asymmetry of the protoplasm itself to account for the P.D. Since appreciable currents are produced, there must be some metabolic activity as a source of energy. The higher normal P.D. in H. ovalis is not due to the higher KCl content of its sap (as earlier suggested by the author) since it persists nearly unchanged when sea water is substituted for sap.     

CALCULATIONS OF BIOELECTRIC POTENTIALS : II. THE CONCENTRATION POTENTIAL OF KCl IN NITELLA

Cells of Nitella have been studied which behave differently from those described in earlier papers. They show unexpectedly large changes in P.D. with certain concentrations of KCl. This is due to the production of action currents (these are recorded at the spot where KCl is applied). A method is given for the separate evaluation of changes of P.D. due to partition coefficients and those due to mobilities. A new amplifier and an improved flowing contact are described.     

THE VARIATION OF ELECTRICAL RESISTANCE WITH APPLIED POTENTIAL : III. IMPALED VALONIA VENTRICOSA

Electrical resistance and polarization were measured during the passage of direct current across a single layer of protoplasm in the cells of Valonia ventricosa impaled upon capillaries. These were correlated with five stages of the P.D. existing naturally across the protoplasm, as follows: 1. A stage of shock after impalement, when the P.D. drops from 5 mv. to zero and then slowly recovers. There is very little effective resistance in the protoplasm, and polarization is slight. 2. The stage of recovery and normal P.D., with values from 8 to 25 mv. (inside positive). The average is 15 mv. At first there is little or no polarization when small potentials are applied in either direction across the protoplasm, nor when very large currents pass outward (from sap to sea water). But when the positive current passes inward there is a sudden response at a critical applied potential ranging from 0.5 to 2.0 volts. The resistance then apparently rises as much as 10,000 ohms in some cases, and the rise occurs more quickly in succeeding applications after the first. When the potential is removed there is a back E.M.F. displayed. Later there is also an effect of such inward currents which persists into the first succeeding outward flow, causing a brief polarization at the first application of the reverse potential. Still later this polarization occurs at every exposure, and at increasingly lower values of applied potentials. Finally there is a "constant" state reached in which the polarization occurs with currents of either direction, and the apparent resistance is nearly uniform over a considerable range of applied potential. 3. A state of increased P.D.; to 100 mv. (inside positive) in artificial sap; and to 35 or 40 mv. in dilute sea water or 0.6 M MgSO₄. The polarization response and apparent resistance are at first about as in sea water, but later decrease. 4. A reversed P.D., to 50 mv. (outside positive) produced by a variety of causes, especially by dilute sea water, and also by large flows of current in either direction. This stage is temporary and the cells promptly recover from it. While it persists the polarization appears to be much greater to outward currents than to inward. This can largely be ascribed to the reduction of the reversed P.D. 5. Disappearance of P.D. caused by death, and various toxic agents. The resistance and polarization of the protoplasm are negligible. The back E.M.F. of polarization is shown to account largely for the apparent resistance of the protoplasm. Its calculation from the observed resistance rises gives values up to 150 mv. in the early stages of recovery, and later values of 50 to 75 mv. in the "constant" state. These are compared with the back E.M.F. similarly calculated from the apparent resistance of intact cells. The electrical capacitance of the protoplasm is shown by the time curves to be of the order of 1 microfarad per cm.² of surface.     

PAP and NT5E inhibit nociceptive neurotransmission by rapidly hydrolyzing nucleotides to adenosine

Background

Acid-sensing ion channels (ASICs) have a significant role in the sensation of pain and constitute an important target for the search of new antinociceptive drugs. In this work we studied the antinociceptive properties of the BM-21 extract, obtained from the sea grass Thalassia testudinum, in chemical and thermal models of nociception in mice. The action of the BM-21 extract and the major phenolic component isolated from this extract, a sulphated flavone glycoside named thalassiolin B, was studied in the chemical nociception test and in the ASIC currents of the dorsal root ganglion (DRG) neurons obtained from Wistar rats.

Results

Behavioral antinociceptive experiments were made on male OF-1 mice. Single oral administration of BM-21 produced a significant inhibition of chemical nociception caused by acetic acid and formalin (specifically during its second phase), and increased the reaction time in the hot plate test. Thalassiolin B reduced the licking behavior during both the phasic and tonic phases in the formalin test. It was also found that BM-21 and thalassiolin B selectively inhibited the fast desensitizing (τ < 400 ms) ASIC currents in DRG neurons obtained from Wistar rats, with a nonsignificant action on ASIC currents with a slow desensitizing time-course. The action of thalassiolin B shows no pH or voltage dependence nor is it modified by steady-state ASIC desensitization or voltage. The high concentration of thalassiolin B in the extract may account for the antinociceptive action of BM-21.

Conclusions

To our knowledge, this is the first report of an ASIC-current inhibitor derived of a marine-plant extract, and in a phenolic compound. The antinociceptive effects of BM-21 and thalassiolin B may be partially because of this action on the ASICs. That the active components of the extract are able to cross the blood-brain barrier gives them an additional advantage for future uses as tools to study pain mechanisms with a potential therapeutic application.     

OBSERVATIONS ON THE INJURIOUS EFFECTS OF HIGH TEMPERATURES ON FROG SKIN

1. The P.D. between the two surfaces of the skin of the frog, when measured under standardized conditions, may be used as a means of studying the phenomena of injury, recovery, and death. Injurious conditions decrease the P.D. 2. According to the severity of the injury produced by temporary exposure to high temperatures, frog skin may show, as judged by this criterion (a) complete recovery, (b) partial recovery, or (c) a progressive change leading to death.     

Inhibitory Actions of Synthesised Polyamine Spider Toxins and Their Analogues on Neuronal Voltage-Activated Calcium Currents

The whole cell variant of the patch-clamp technique was used to investigate the actions of polyamine spider toxins and their analogues on high voltage-activated Ca²⁺ currents. The actions of synthesised FTX (putative natural toxin from the American funnel web spider), sFTX-3.3, Orn-FTX-3.3 and Lys-FTX-3.3 (synthetic analogues of FTX) were studied using cultured dorsal root ganglion neurones from neonatal rats, C2D7 cells (HEK293 cells stably coexpressing recombinant human N-type voltage-activated Ca²⁺ channel, α_1B-1-α_2bδβ_1b subunits) and freshly isolated cerebellar Purkinje neurones. In dorsal root ganglion neurones, sFTX-3.3 (10 μM) inhibited high voltage-activated Ca²⁺ currents evoked by depolarisations to 0 mV from a holding potential of −90 mV. Partial overlap in Ca²⁺ current sensitivity to the polyamine sFTX-3.3 and the peptide spider toxin ω-Aga IVA was observed. However, evidence also suggests sFTX-3.3 and ω-Aga IVA do not show complete pharmacological overlap and that distinct parts of the Ca²⁺ current are sensitive to one of two inhibitors. The arginine group on sFTX-3.3 appears to be important for its inhibitory action on Ca²⁺ currents, because analogues where this amino acid was replaced with either ornithine (Orn-FTX-3.3) or lysine (Lys-FTX-3.3) were relatively inactive at concentrations below 1 mM. Synthesised FTX (100 μM) was inactive as an inhibitor of Ca²⁺ currents recorded from dorsal root ganglion and only produced modest effects in Purkinje neurones and C2D7 cells. At a concentration of 1 mM, nonselective actions were observed that indicated that synthesised FTX and sFTX-3.3 could reversibly inhibit both N- and P-type Ca²⁺ channels equally well. In conclusion, the potency of polyamines as nonselective inhibitors of Ca²⁺ channels is in part determined by the presence of a terminal arginine, and this may involve an interaction between terminal guanidino groups with Ca²⁺ binding sites.     

Macroscopic Na+ Currents in the “Nonconducting” Shaker Potassium Channel Mutant W434F

C-type inactivation in Shaker potassium channels inhibits K⁺ permeation. The associated structural changes appear to involve the outer region of the pore. Recently, we have shown that C-type inactivation involves a change in the selectivity of the Shaker channel, such that C-type inactivated channels show maintained voltage-sensitive activation and deactivation of Na⁺ and Li⁺ currents in K⁺-free solutions, although they show no measurable ionic currents in physiological solutions. In addition, it appears that the effective block of ion conduction produced by the mutation W434F in the pore region may be associated with permanent C-type inactivation of W434F channels. These conclusions predict that permanently C-type inactivated W434F channels would also show Na⁺ and Li⁺ currents (in K⁺-free solutions) with kinetics similar to those seen in C-type-inactivated Shaker channels. This paper confirms that prediction and demonstrates that activation and deactivation parameters for this mutant can be obtained from macroscopic ionic current measurements. We also show that the prolonged Na⁺ tail currents typical of C-type inactivated channels involve an equivalent prolongation of the return of gating charge, thus demonstrating that the kinetics of gating charge return in W434F channels can be markedly altered by changes in ionic conditions.     

Kinetic Model for NS1643 Drug Activation of WT and L529I Variants of Kv11.1 (hERG1) Potassium Channel

Congenital and acquired (drug-induced) forms of the human long-QT syndrome are associated with alterations in Kv11.1 (hERG) channel-controlled repolarizing I_Kr currents of cardiac action potentials. A mandatory drug screen implemented by many countries led to a discovery of a large group of small molecules that can activate hERG currents and thus may act as potent antiarrhythmic agents. Despite significant progress in identification of channel activators, little is known about their mechanism of action. A combination of electrophysiological studies with molecular and kinetic modeling was used to examine the mechanism of a model activator (NS1643) action on the hERG channel and its L529I mutant. The L529I mutant has gating dynamics similar to that of wild-type while its response to application of NS1643 is markedly different. We propose a mechanism compatible with experiments in which the model activator binds to the closed (C3) and open states (O). We suggest that NS1643 is affecting early gating transitions, probably during movements of the voltage sensor that precede the opening of the activation gate.     

STIMULATION BY COLD IN NITELLA

Sudden local chilling causes action currents to be set up in Nitella and in Chara, an effect which does not follow gradual local chilling. This may be due to a partial solidification of the non-aqueous protoplasmic surfaces which makes them susceptible to rupture by the protoplasmic streaming. This movement continues usually for several minutes after the chilling, whereas if stimulation occurs at all it occurs immediately on chilling. It is found that a chilled spot is much more sensitive to mechanical stimulation than is a spot at room temperature. Chilling is accompanied by a rise of resistance, a lowered rate of recovery following stimulation, and usually by a falling off in the magnitude of the action curve.     

Ionic currents during the action potential in the molluscan neurone with the self-clamp technique

The ionic currents during the action potential in the F1 neurone of Helix aspersa were investigated, using the Self-Clamp Technique. A spontaneous action potential was recorded and then replayed, both in its direct and in its inverted form, to the same cell in voltage clamp and in control conditions. Under various experimental conditions such as treatment with the specific ionic channels blockers tetrodotoxin, lanthanum, 4-aminopyridine or tetraethylammonium, as well as low sodium and low calcium external media, the single ionic currents were detected by stimulating the membrane with the direct pulse only. The Self-Clamp Technique allowed the measuring of the following parameters, in their real time course during the action potential: a) the total action currents; b) the pharmacologically blocked ionic components; c) the ionic components which remained insensitive to the agents used (residual currents). These data were compared with those obtained by applying conventional rectangular pulses in voltage clamp. The membrane capacity was measured with the Self-Clamp Technique and the recorded currents were normalized assuming a specific capacity of 4 μF/cm². The isolated ionic components were directly compared with the total action currents to evaluate the degree to which blockage was complete. The electric charge transported by each ionic specimen was evaluated as well as the individual ionic amounts. The sodium influx was 3.18 ± 0.55 pM/cm² per impulse (9 cells), calcium influx 1.03 ± 0.37 pM/cm² per impulse (10 cells). A value of 6.37 ± 1.03 pM/cm² per impulse was found for the potassium outflux, with a probable overestimation of about 1 pM/cm² per impulse (9 cells).