Gating currents and charge movements in excitable membranes.
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Animals↗
Biomedical subjects
W Almers
Publications and source records attributed to W Almers.
Local anesthetics and excitation-contraction coupling in skeletal muscle. Effects on a Ca++-channel [proceedings].
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Anesthetics, Local↗
The voltage dependence of membrane capacity.
1. Membrane capacity of sartorius muscle fibres has been measured at membrane potentials between -200 and +50 mV. Within this potential range the capacity is not independent of potential. Dielectric saturation is present at large negative and at positive internal potentials, indicating the presence in the membrane of permanent dipoles or movable charges. 2. In normally polarized fibres there is a sharp peak in the capacity-potential relation of about -50 mV; the capacity at this peak is 50% larger than the capacity at -90 mV. 3. In depolarized fibres this sharp peak of capacity is not present. Over the range -200 to +50 mV the capacity variation is about 10% with a broad maximum at about -80 mV. 4. The dielectric behaviour of muscle membrane is most simply explained by postulating two species of permanent dipoles or mobile charges: Charge 1 present in normally polarized fibres, but neutralized or immobilized in depolarized fibres; Charge 2 present in both polarized and depolarized fibres. The distribution of Charge 1 is more steeply voltage-dependent than is the distribution of Charge 2. 5. Movement of Charge 1 from one fully saturated configuration to the other involves a charge transfer across the membrane of between 20 and 30 nC/muF. Movement of Charge 2 in depolarized fibres requires a similar transfer of charge.
Animals↗
Charge movement in the membrane of striated muscle.
1. Non-linear polarization currents apparently due to permanent dipoles or mobile charges in the membrane can be measured by appropriate comparison of the transient currents required to produce small and large steps of membrane potential. Integration of these transient polarization currents estimates the charge transfer associated with the movement of membrane dipoles or charges. 2. Depolarization from -100 to 0 mV requires a charge transfer of 35 nC/muF in addition to the charge transfer predicted by linear extrapolation of the charge required for a small depolarization from -100 mV. Depolarizations of varying size give a charge-voltage relation which is sigmoid saturating beyond o mV and with a midpoint at about -50 mV. The ratnged depolarization reduces or removes charge movement detected by comparing currents for small and large voltage steps from -100 mV (Charge 1). However in depolarized fibres comparison of currents from a small potential step at +40 mV and a large hyperpolarizing potential step from -20 mV reveals large movements of a second charge (Charge 2). Movement of Charge 2 is less steeply dependent on voltage than movement of Charge 2 both in magnitude and in rate. 4. In size and voltage dependence these two kinds of charge movement correspond to measured voltage dependence of capacity in normally polarized and depolarized fibres (Adrian & Almers, 1976).
Animals↗
Effects of tetracaine on displacement currents and contraction of frog skeletal muscle.
The kinetics of mechanical activation of intact fibres were examined with a voltage-clamp technique. Tetracaine (2 mM) increases fifteen- to seventyfold the time required to produce a just visible contraction by cell membrane depolarization. 2. Displacement currents thought to be related to contractile activation remain in 2 mM tetracaine. Their characteristics are virtually identical to those found in the absence of the drug. Displacement currents also remain in fibres immobilized by treatment with 10 mM formaldehyde. 3. Despite its effect on contraction of intact fibres, tetracaine does not diminish contraction tension when Ca is applied directly to the contractile proteins of 'skinned' muscle fibres. The sensitivity of the myofilaments to Ca2+ also remains undiminished. 4. When acting on intact fibres the drug must therefore inhibit Ca2+-release from the sarcoplasmic reticulum. It is estimated that 2 mM tetracaine diminishes more than tenfold the capacity for Ca2+-release in response to cell membrane depolarization.5. If muscle displacement currents represent events linking depolarization to Ca2+-release, then tetracaine must be able to block the release without affecting the potential-sensing portion of the release regulating mechanism. 6. Further experiments on skinned fibres show that tetracaine blocks or greatly diminishes caffeine contractions, but that Cl-induced contractions of normal amplitude are still possible.
Animals↗
Differential effects of tetracaine on delayed potassium channels and displacement currents in frog skeletal muscle.
1. Delayed K+-currents and displacement currents were studied with a voltage-clamp technique. 2. In normal fibres, the conductance of the delayed channel grows e-fold per 3 millivolts at sufficiently negative potentials and reaches a limiting value of 2-10 m-mho/cm2 (mean 5-8 m-mho/cm2) at positive potentials. Adding tetracaine (2 mM) reduces the limiting conductance, shifts the voltage-dependence of the delayed channel to +25 mV more positive potentials and slows the kinetics fourfold. 3. By contrast, the displacement currents are virtually unaltered by 2 mM tetracaine. Their voltage-dependence is shifted by less than 5 mV and their kinetics are unaffected. 4. Tetraethylammonium ions (TEA) are known to slow the kinetics of delayed K+-channels fivefold but fail, like tetracaine, to change the kinetics of the displacement currents. 5. Both tetracaine and TEA have thus large effects on the 'gating' of the delayed channel, yet little or none on the displacement currents. This suggests that the displacement currents in skeletal muscle are for the most part unrelated to the opening and closing of delayed channels. It is estimated that 'gating' the delayed channel in muscle may require no more than 1 or 2% of the observed charge displacement.
Animals↗
Some dielectric properties of muscle membrane and their possible importance for excitation-contraction coupling.
Voltage-clamp studies of polarization currents have shown the presence of polar molecules in the membrane of skeletal muscle. Their high dipole moment and favorable transition potential suggest that they may be "gating molecules" which serve to regulate some membrane potential-dependent physiological function. Experiments with the drugs tetrodotoxin and tetracaine show that these polar molecules are unlikely to play a role in regulating the sodium and potassium channels responsible for electrical excitation. Instead, they may regulate calcium release from the sarcoplasmic reticulum, which also depends steeply on the cell membrane potential. Schneider and Chandler's hypothesis whereby calcium release from the sarcoplasmic reticulum is regulated by voltage-sensitive "gating molecules" in the tubule membrane is still unproven, but remains attractive and plausible.
Animals↗
Observations on intramembrane charge movements in skeletal muscle.
Using signal-averaging techniques, one can record small membrane currents which remain even after blockage of the ionic currents which accompany electrical excitation in muscle. These residual currents probably represent the reorientation of charged molecules inside the membrane in response to a change in membrane potential. Two operationally separable types of intramembrane charge movement in muscle are described, one of which may play a role in excitation-contraction coupling. Studies of tetrodotoxin binding to muscle indicate that "sodium gating current" is unlikely to contribute significantly to either type of charge movement.
Action Potentials↗
Tetrodotoxin binding to normal depolarized frog muscle and the conductance of a single sodium channel.
1. We have examined the binding of tritium-labelled and unlabelled tetrodotoxin to frog twitch muscle. Bio-assay as well as radioisotope experiments show a saturable component of tetrodotoxin binding with a binding capacity of about 22 p-mole/g wet wt., and a dissociation constant of about 5 nM. 2. If the observed uptake of tetrodotoxin by muscles represents one-to-one binding of the drug to sodium channels, the channel density is about 380 channels/mum2 of a muscle fibre's surface membrane. On the basis of this result and electrical measurements of sodium conductance in frog muscle, we calculate that the conductance of a single sodium channel is of the order of 10(-12) reciprocal ohms. This is one to two orders of magnitude less than previous estimates. 3. We have looked for an effect of membrane depolarization on saturable tetrodotoxin binding, and have found none. This suggests that there is little molecular interaction between the "gating" portion of the sodium channel molecule, and that which binds tetrodotoxin.
Action Potentials↗
Membrane capacity measurements on frog skeletal muscle in media of low ion content.
1. A voltage-clamp technique for the measurement of effective membrane capacity is described and its theoretical basis given in an Appendix. Unlike most other methods used so far, the present technique does not depend on the assumption of a particular equivalent circuit for muscle membrane.2. With this technique, we have re-examined the effect of media of low ion content on the effective membrane capacity C(eff). In fibres where potassium conductance is blocked by Rb, C(eff) failed to show any appreciable dependence on ionic strength (between 0.015 and 0.29 M) or conductivity (between 0.86 and 13.4 mmho/cm) of the bathing medium.3. Low conductivity can reduce C(eff) in fibres of moderately high K-conductance. However, in all cases explored here, C(eff) had values well above the 2 muF/cm(2) found in glycerol-treated fibres, indicating that passive spread of potential displacements from surface into the tubular system could still occur.4. The changes in C(eff) observed under conditions of moderately high K-conductance are explicable as a result of potential decrements in the transverse tubules, which would be expected when the wall conductance there is high, and the conductivity of the tubule lumen is low.
Animals↗
Measurement of membrane capacity in skeletal muscle.
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Animals↗
Potassium conductance changes in skeletal muscle and the potassium concentration in the transverse tubules.
1. When one hyperpolarizes a muscle fibre by passing electric current, the K conductance declines with time. Voltage-clamp experiments on frog sartorius muscle fibres showed that two components contribute to this decline.2. A rapid component operates when the fibre is hyperpolarized to potentials more negative than -120 mV. Decline by this mechanism is reversed completely within 200 msec. The large effect of temperature on the kinetics of this process indicates that it represents a time-dependent membrane permeability change.3. A slow component operates also at less negative potentials. Recovery at -65 mV takes place with half-times of about 0.4 sec. The Q(10) for the rate of recovery is 1.3, indicating that this process is diffusion limited.4. After prolonged hyperpolarization to potentials positive to -120 mV, membrane current at the resting potential is outward and persists for several seconds. At that time, the potential measured in the absence of membrane current is shifted in the negative direction by 3-5 mV.5. This shift and the time course of currents near the resting potential after hyperpolarization as well as the Q(10) of 1.3 indicate that the slow process is due to changes in tubular K concentration and not to a time-dependent membrane permeability change.6. At potentials less negative than -120 mV, tubular depletion can satisfactorily account for the decline of K conductance. At more negative potentials, the decline appears to be due to both depletion and a permeability change.
Animals↗
The decline of potassium permeability during extreme hyperpolarization in frog skeletal muscle.
1. The voltage-clamp technique was used to separate the effects of K depletion in the T-system from the decline in K permeability during hyperpolarization, and to characterize the time- and voltage-dependence of the latter.2. K permeability due to the inward rectifier can be described as being proportional to a parameter which diminishes when the membrane is hyperpolarized beyond -120 mV. The parameter obeys first-order kinetics. At 24 degrees C, it can change with a time constant of 49 msec at -150 mV and 25 msec at -65 mV. At -200 mV the fall in membrane conductance due to the permeability change is to 30% of its initial value. The Q(10) for the rate of conductance change at that potential is about 2.8.3. It is estimated that K inward current can lower the average K concentration in the T-system by more than 50%, and that, on the average, the space enclosed by the T-system should be less than 0.8% of the fibre volume. Assuming the T-system space to be 0.3% of the fibre volume, it is calculated that on the average, and during hyperpolarization to about -150 mV, no more than 20% of the initial current should flow across the surface membrane.
Animals↗
Dihydropyridine receptors in muscle are voltage-dependent but most are not functional calcium channels.
1,4-Dihydropyridines are a new class of compounds believed to bind specifically and with high affinity to voltage-dependent calcium channels. They may be the first example of a ligand of use in the extraction and purification of the Ca channel. Although Ca channels and dihydropyridine receptors are found in many tissues, the richest and most convenient source is skeletal muscle. Functionally, 1,4-dihydropyridines such as nifedipine and nitrendipine block Ca channels; this effect is believed to form the basis for their clinical importance as Ca antagonists in relaxing vascular smooth muscle. But where currents through Ca channels can be measured directly, the block has required 100-1,000 times higher concentrations of dihydropyridine than necessary for the saturation of dihydropyridine binding sites. This discrepancy has remained unresolved because the study of pharmacological effects on Ca channels has required intact cells, while it has been difficult to investigate binding in other than cell-free preparations. Here we describe a method for measuring dihydropyridine binding to intact skeletal muscle and we compare our results with voltage-clamp measurements of Ca-channel block. We conclude that less than a few per cent of the binding sites in skeletal muscle represent functional Ca channels, contrary to general belief.
Animals↗
Currents through the fusion pore that forms during exocytosis of a secretory vesicle.
Exocytosis, or the fusion of cytoplasmic vesicles with the cell membrane, occurs in nearly all eukaryotic cells, but its mechanism is not understood. Morphological and electrophysiological studies have suggested that membrane fusion begins with the formation of a 'fusion pore', a narrow channel across the closely adjacent membranes of vesicle and cell that forms the first connection of the vesicle lumen with the cell exterior and later dilates to allow release of vesicle contents. We used the patch clamp technique to study exocytosis of single giant secretory vesicles in mast cells of beige mice. The first opening of the fusion pore was found to generate a brief current transient, whose size and direction indicated an initial pore conductance of about 230 pS and a lumen-positive vesicle membrane potential. In time-resolved a.c. admittance measurements, the pore conductance was found to increase to much larger values within milliseconds, as if the pore dilated soon after opening. We conclude that the earliest fusion event may be the formation of a structure similar to an ion channel. Its conductance is of the same order of magnitude as that of a single gap junction channel, the only other known channel that spans two membranes.
Animals↗