Vaseline-gap Method Research Articles

Effects of membrane depolarization and changes in extracellular [K+] on the Ca2+ transients of fast skeletal muscle fibers. Implications for muscle fatigue

Repetitive activation of skeletal muscle fibers leads to a reduced transmembrane K+ gradient. The resulting membrane depolarization has been proposed to play a major role in the onset of muscle fatigue. Nevertheless, raising the extracellular K+ ( {text{K}}_{text{o}}^{ + } ) concentration ( [ {text{K}}^{ + } ]_{text{o}} ) to 10 mM potentiates twitch force of rested amphibian and mammalian fibers. We used a double Vaseline gap method to simultaneously record action potentials (AP) and Ca2+ transients from rested frog fibers activated by single and tetanic stimulation (10 pulses, 100 Hz) at various [ {text{K}}^{ + } ]_{text{o}} and membrane potentials. Depolarization resulting from current injection or raised [ {text{K}}^{ + } ]_{text{o}} produced an increase in the resting [Ca2+]. Ca2+ transients elicited by single stimulation were potentiated by depolarization from −80 to −60 mV but markedly depressed by further depolarization. Potentiation was inversely correlated with a reduction in the amplitude, overshoot and duration of APs. Similar effects were found for the Ca2+ transients elicited by the first pulse of 100 Hz trains. Depression or block of Ca2+ transient in response to the 2nd to 10th pulses of 100 Hz trains was observed at smaller depolarizations as compared to that seen when using single stimulation. Changes in Ca2+ transients along the trains were associated with impaired or abortive APs. Raising [ {text{K}}^{ + } ]_{text{o}} to 10 mM potentiated Ca2+ transients elicited by single and tetanic stimulation, while raising [ {text{K}}^{ + } ]_{text{o}} to 15 mM markedly depressed both responses. The effects of 10 mM {text{K}}_{text{o}}^{ + } on Ca2+ transients, but not those of 15 mM {text{K}}_{text{o}}^{ + } , could be fully reversed by hyperpolarization. The results suggests that the force potentiating effects of 10 mM {text{K}}_{text{o}}^{ + } might be mediated by depolarization dependent changes in resting [Ca2+] and Ca2+ release, and that additional mechanisms might be involved in the effects of 15 mM {text{K}}_{text{o}}^{ + } on force generation.

Differential contribution of Na +-K + pump and K + conductance to the post-tetanic hyperpolarization of the subtypes of tetrodotoxin-resistant C-fibers in the isolated bullfrog sciatic nerve

The post-tetanic hyperpolarizations (PTHPs) of the fast and slow tetrodotoxin-resistant (TTX-R) C-fibers were recorded from isolated bullfrog sciatic nerves using a modified vaseline gap method. Under control solutions, the monophasic fast and slow TTX-R C-fibers compound action potentials (C-CAPs) increased in amplitude up to 133.9±17.9% (mean±SD; n=9) and 167.0±41.5% ( n=7), respectively, shortly after tetanic stimulation. The recovery time constants of the fast and slow ones were 66.5 and 101.2 s, respectively. Significant differences between the rates of amplitude augmentation of the fast and slow TTX-R C-CAPs were found for the most pairs of measured points ( P<0.05 or P<0.01). After application of ouabain solutions, the augmentation of the fast TTX-R C-CAP was inhibited to an insignificant level, while that of the slow TTX-R C-CAP remained significant with the recovery time constant being 90.9 s. After additional application of TEA, the post-tetanic augmentation of the slow TTX-R C-CAP was blocked. These results suggest that Na +-K + pump contributes to the PTHPs of the fast and slow TTX-R C-fibers and that K + conductance contributes only to the PTHP of the slow TTX-R C-fibers, indicating differential modulation of the firing rate of each TTX-R C-fibers group.

Evidence for the involvement of Na +-K + pump and K + conductance in the post-tetanic hyperpolarization of the tetrodotoxin-resistant C-fibers in the isolated bullfrog sciatic nerve

The post-tetanic hyperpolarizations (PTHPs) were recorded from the isolated bullfrog sciatic nerves using a modified vaseline gap method. Repetitive stimulation of tetrodotoxin-resistant (TTX-R) C-fibers produced frequency- and activity-dependent PTHPs. The ouabain solutions (0.1 mM), the solutions (0.1 mM ouabain+2 mM tetraethylammonium (TEA)) and 5 mM TEA solutions reduced the PTHP amplitude to 56.2±10.7% (mean±SD, n=9), to 37.0±12.3% ( n=8), to 76.0±10.9% ( n=5) of the control value, respectively. Each pair of the three solutions revealed a significant difference in their effects on the PTHP ( P<0.01). Furthermore, the Li–Ringer solutions and the solutions (Li–Ringer+2 mM TEA) reduced the PTHP amplitude as well, thus resembling ouabain effects. These results strongly suggest that the repetitive stimulation of TTX-R C-fibers activates the membrane Na +-K + pump resulting in a major part of the PTHP and induces TEA-sensitive K + conductance composing a minor part of the PTHP, which might modulate repetitive firing of TTX-R C-fibers.

Tetrodotoxin-resistant Na + spikes of C fibers have at least two subtypes in the isolated bullfrog sciatic nerve

The C fiber compound action potentials (C-CAPs) were studied for their properties with the vaseline gap method from the isolated bullfrog sciatic nerve. Two components (the fast and the slow C-CAPs) were found to be Na + dependent and tetrodotoxin-resistant. The conduction velocities of the fast and the slow C-CAPs were 0.61±0.06 m/s ( n=13) and 0.42±0.05 m/s ( n=8), respectively; their time constants were 11.4±1.7 ms and 16.3±1.7 ms, respectively, with both parameters being significantly different at P<0.01. The slow C-CAP had about 1.3 times higher threshold than that of the fast C-CAP, with a significant difference ( P<0.01). They showed differential sensitivity to lidocaine and Cd 2+. Capsaicin reduced the amplitudes of both fast and slow C-CAPs, but not abolished. These findings indicate that C fibers have at least two subtypes with different properties.

Pandinus imperator scorpion venom blocks voltage-gated potassium channels in nerve fibers

We have examined the effects of venom from the scorpion Pandinus imperator on the membrane currents of voltage-clamped frog myelinated nerve fibers using the Vaseline-gap method. Crude venom, applied externally in concentrations from 50 to 500 micrograms/ml, selectively blocked the voltage-gated potassium currents without affecting nodal sodium currents or resting conductances. Block of potassium channels by Pandinus venom was highly dependent on the membrane voltage, being greater at negative potentials than at positive potentials. The blocking effects of Pandinus venom were irreversible on the time scale of our experiments; however, even high concentrations of venom failed to block potassium currents completely at positive potentials. These results suggest that Pandinus venom contains a component(s) that interacts specifically and strongly with a subpopulation of axonal potassium channels.

Passive electrical properties and voltage dependent membrane capacitance of single skeletal muscle fibers.

The passive membrane capacitance and conductance of isolated single muscle fibers were investigated using a vaseline gap method. The results obtained with this method are consistent with those obtained using the microelectrode technique. It was confirmed that the membrane capacitance of skeletal muscle consisted of a large capacitance of tubular membrane (7-10 microF/cm2) and a much smaller capacitance of surface membrane (1-2 microF/cm2). The relative time constants of these two components vary from one sample to another, resulting in one time and two time constant behaviors. Secondly, the capacitance of isolated skeletal muscle fibers was investigated during hyper- and depolarizing pulses, using the transient bridge technique with the vaseline gap method. Measurements were performed at two frequencies, i.e. 500 Hz and 20 kHz. It was found that the membrane capacitance increased by 15-20% with depolarizations. The voltage dependent membrane capacitance was not affected by the addition of tetrodotoxin in bathing solution blocking sodium current and muscle contraction. Also, blocking both Na and K current did not have an appreciable effect on the non-linear behavior of membrane capacitance. The origin of voltage dependent capacitance in muscle membrane appears to be distributed among several non-linear ionic processes such as Na and K currents and the flux of Ca and Cl ions and their accumulation.

The interaction of potassium with the activation of anomalous rectification in frog muscle membrane.

1. Inward rectification of frog muscle membrane was analysed with the Vaseline gap method. 2. Hyperpolarization, under voltage clamp, produced inward potassium currents, which had a component that activated with a time constant, tau K. 3. The activation time constant tau K of the inward potassium currents was voltage dependent. For a given external potassium concentration, the time constant was maximal for potentials near the potassium equilibrium potential, EK. 4. The potassium chord conductance gK, had a sigmoidal voltage dependency, increasing initially e-fold per 11.6 mV of hyperpolarization. 5. When the internal potassium concentration was fixed, raising external potassium induced a shift of the tau K-V and the gK-V relations in the positive direction along the voltage axis. That shift was comparable to the change in EK. 6. No shift of the tau K-V and the gK-V relations was observed when the internal potassium was reduced from 150 to 50 mM. 7. Changes of internal sodium concentration between 5 and 100 mM did not significantly effect the magnitude of inward rectification.