The effects of tetracaine on charge movement in fast twitch rat skeletal muscle fibres.

The effects of the ryanodine receptor (RyR) antagonists ryanodine and daunorubicin on the kinetic and steady-state properties of intramembrane charge were investigated in intact voltage-clamped frog skeletal muscle fibers under conditions that minimized timedependent ionic currents. A hypothesis that RyR gating is allosterically coupled to configurational changes in dihydropyridine receptors (DHPRs) would predict that such interactions are reciprocal and that RyR modification should influence intramembrane charge. Both agents indeed modified the time course of charging transients at 100-200-p.M concentrations. They independently abolished the delayed charging phases shown by q~ currents, even in fibers held at fully polarized, -90-mV holding potentials; such waveforms are especially prominent in extracellular solutions containing gluconate. Charge movements consistently became exponential decays to stable baselines in the absence of intervening inward or other time-dependent currents. The steady-state charge transfers nevertheless remained equal through the ON and the OFF parts of test voltage steps. The charge-voltage function, Q(VT), shifted by ~+ 10 mV, particularly through those test potentials at which delayed q~ currents normally took place but retained steepness factors (k ~ 8.0 to 10.6 mV) that indicated persistent, steeply voltage-dependent q~ contributions. Furthermore, both RyR antagonists preserved the total charge, and its variation with holding potential, Qma~(VH), which also retained similarly high voltage sensitivities (k ~ 7.0 to 9.0 mV). RyR antagonists also preserved the separate identities of qv and qa species, whether defined by their steady-state voltage dependence or inactivation or pharmacological properties. Thus, tetracaine (2 mM) reduced the available steady-state charge movement and gave shallow Q(VT) (k --~ 14 to 16 mV) and Qma~(VH) (k 14 to 17 mV) curves characteristic of q~ charge. These features persisted with exposure to test agent. Finally, q~ charge movements showed steep voltage dependences with both activation (k ~ 4.0 to 6.5 mV) and inactivation characteristics (k ~ 4.3 to 6.6 mV) distinct from those shown by the remaining q~ charge, whether isolated through differential tetracaine sensitivities, or the full approximation of charge-voltage data to the sum of two Boltzmann distributions. RyR modification thus specifically alters qx kinetics while preserving the separate identities of steady-state qf~ and q~ charge. These findings permit a mechanism by which transverse tubular voltage provides the primary driving force for configurafional changes in DHPRs, which might produce q~ charge movement. However, they attribute its kinetic complexities to the reciprocal allosteric coupling by which DHPR voltage sensors and RyR-Ca 2 § release channels might interact even though these receptors reside in electrically distinct membranes. RyR modification then would still permit tubular voltage change to drive net q~ charge transfer but would transform its complex waveforms into s...

Section: Discussionmentioning

confidence: 99%

Kinetic isoforms of intramembrane charge in intact amphibian striated muscle.

Huang

1996

“…This applies to some amphibian preparations using cut fibers (Melzer et al, 1986;cf. Csernoch et al, 1988), and on occasion to mammalian muscle (Lamb, 1986;Hollingworth et al, 1990;cf. Hollingworth and Marshall, 1981;Simon and Beam, 1985).…”

Section: Discussionmentioning

confidence: 99%

Separation of intramembrane charging components in low-calcium solutions in frog skeletal muscle.

Huang

1991

A B S T R A C T The inactivation of charge movement components by small (-100 to -70 mV) shifts in holding potential was examined in voltage-clamped intact amphibian muscle fibers in low [Ca2+], Mg2+-containing solutions. The pulse protocols used both large voltage excursions and smaller potential steps that elicited prolonged (q~) transients. Charge species were distinguished through the pharmacological effects of tetracaine. These procedures confirmed earlier observations in cut fibers and identified the following new properties of the q~ charge. First, %, previously defined as the tetracaine-sensitive charge, is also the component primarily responsible for the voltage-dependent inactivation induced by conditions of low extracellular [Ca2+]. Second, this inactivation separates a transient that includes a "hump" component and which has kinetics and a voltage dependence distinct from the monotonic decay that remains. Third, qv, previously associated with delayed charge movements, can also contribute significant charge transfer at early times. These findings suggest that the parallel inhibition of calcium signals and charge movements reported in low [Ca z+] solutions arises from influences on q~ charge (Brum et al., 1988a, b). They also reconcile reports that implicate tetracainesensitive (q~) charge in excitation-contraction coupling with evidence that early intramembrane events are also involved in this process . Finally, they are relevant to hypotheses of possible feedback or feed-forward roles of q~ in excitation--contraction coupling,

“…This is why all the investigators who observed a suppression of charge movement by tetracaine (Huang, 1982;Hui, 1983a;Lamb, 1986;Hollingworth et al, 1990) did not mention the reversibility of the effect. Vergara and Caputo (1983), on the other hand, specifically mentioned that the suppression of charge movement in cut fibers by > 0.5 mM tetracaine is only partially reversible.…”

Section: Reversibility Of the Suppression Of Charge Movement By Tetramentioning

confidence: 99%

Separation of Q beta and Q gamma charge components in frog cut twitch fibers with tetracaine. Critical comparison with other methods.

Hui

Chen

1992

Charge movement was measured in frog cut twitch fibers with the double Vaseline-gap technique. 25 ~M tetracaine had very little effect on the maximum amounts of Qo and Q~ but slowed the kinetics of the I~ humps in the ON segments of TEST-minus-CONTROL current traces, giving rise to biphasic transients in the difference traces. This concentration of tetracaine also shifted F~ 3.7 (SEM 0.7) mV in the depolarizing direction, resulting in a difference Q-V plot that was bell-shaped with a peak at ~-50 mV. 0.5-1.0 mM tetracaine suppressed the total amount of charge. The suppressed component had a sigmoidal voltage distribution with V = -56.6 (SEM 1.1) mV, k = 2.5 (SEM 0.5) mV, and qmax/Cm = 9.2 (SEM 1.5) nC/~F, suggesting that the tetracaine-sensitive charge had a steep voltage dependence, a characteristic of the Q~ component. An intermediate concentration (0.1-0.5 raM) of tetracaine shifted V~ and partially suppressed the tetracaine-sensitive charge, resulting in a difference Q-V plot that rose to a peak and then decayed to a plateau level. Following a TEST pulse to >-60 mV, the slow inward current component during a post-pulse to ~ -60 mV was also tetracaine sensitive. The voltage distribution of the charge separated by tetracaine (method 1) was compared with those separated by three other existing methods: (a) the charge associated with the hump component separated by a sum of two kinetic functions from the ON segment of a TEST-minus-CONTROL current trace (method 2), (b) the steeply voltage-dependent component separated from a Q-V plot of the total charge by fitting with a sum of two Boltzmann distribution functions (method 3), and (c) the sigmoidal component separated from the Q-v plot of the final OFF charge obtained with a two-pulse protocol (method 4). The steeply voltagedependent components separated by all four methods are consistent with each other, and are therefore concluded to be equivalent to the same Q~ component. The Address reprint requests to Dr.