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

Abstract

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.

Kinetic isoforms of intramembrane charge in intact amphibian striated muscle.

“…The charge movements remained reduced in amplitude, exponential in form; and gradually increased with depolarization as expected of the % species. Table IV demonstrates Bohzmann parameters describing intramembrane charge in 2 mM tetracaine 525 HUANG that agreed with independent determinations for q~ charge derived from minimizations to the sum of two Boltzmann functions in both the present (see Table II) and earlier studies Hui and Chen, 1992). Neither ryanodine nor daunorubicin altered these values.…”

“…This gave q~ parameters (Qna• ~ 11 nC p~F-l; V* ~ -54 mV, k -~ 4.2 mV) that agreed with the results of permitting both q~ and qv parameters to be free variables (Table II) when RyR antagonists were absent. This independent check for the pharmacological fractionation of intramembrane charge confirmed earlier tests (Hui and Chen, 1992) and further identifies the steeper of the two voltage dependences with a discrete q~ charge. Ryanodine treatment slightly decreased the q~ voltage dependence but to a value (k -~ 5.6-6.4 mV) nevertheless distinct from that of the q~ system (k ~ 14.0-16.0 mV).…”

“…They further confirmed that any changes in q~ charge were too small to post any action of tetracaine on other than q~ charge. Hui and Chen (1992) also established that tetracaine concentrations of >0.5-1.0 mM were required to accomplish such full separations. Scrutinies of both fiber stability and cable constants confirm that use of 2 mM tetracaine permitted viable and stable intact fiber preparations in the present experiments.…”

“…Nevertheless, it provides a consistent empirical charge separation testable against a wide range of independent criteria. Thus, a rigorous comparison of results from (I) defining tetracaine-sensitive charge as q~ and tetracaine resistant charge as % (Huang, 1982); against (2) mathematical techniques based on the original kinetic definitions of q~ and q~ in "on" transients (Hui, 1983); (3) fitting charge-voltage functions to the sum of two independent Boltzmann distributions ; and (4) kinetic qv isolations after partial repolarizations to around -60 mV (see Hui and Chandler, 1991), all yielded quantitatively consistent charge separations (Hui and Chen, 1992, Table III). They further confirmed that any changes in q~ charge were too small to post any action of tetracaine on other than q~ charge.…”

“…These values additionally confirmed the pharmacological analyses described below (cf. Hui and Chen, 1992). When applied to data from fibers exposed to RyR antagonists, the successive iterations yielded progressively diminishing values of one of the Qma~ terms, increasingly positive values of its corresponding transition potential, V*, and increasing values of the standard error of the slope factor k. This resembles recent reports for gluconate-containing solutions (Jong et al, 1995), the similar convergences of which toward single Boltzmann functions were attributed to the smaller proportion of q~ charge or closer values of V* shown by q~ and qv in gluconate-treated fibers.…”