SEPARATION OF Q-BETA AND Q-GAMMA CHARGE COMPONENTS IN FROG CUT TWITCH FIBERS WITH TETRACAINE - CRITICAL COMPARISON WITH OTHER METHODS

Charge movement was measured in frog cut twitch fibers with the double Vaseline-gap technique. 25-mu-M tetracaine had very little effect on the maximum amounts of Q(beta) and Q(gamma) but slowed the kinetics of the I(gamma) humps in the 0 segments of TEST-minus-CONTROL current traces, giving rise to biphasic transients in the difference traces. This concentration of tetracaine also shifted V(gamma)BAR 3. (SEM 0.7) mV in the depolarizing direction, resulting in a difference Q-V plot that was bell-shaped with a peak at approximately -50 mV. 0.5-1.0 mM tetracaine suppressed th total amount of charge. The suppressed component had a sigmoidal voltage distribution with VBAR = 56.6 (SEM 1. 1) mV, k = 2.5 (SEM 0. 5) mV, and q(max)/C(m) = 9.2 (SEM 1.5) nC/mu-F, suggesting that the tetracaine-sensitive charge had a steep voltage dependence, a characteristic of the Q(gamma) component. An intermediate concentration (0.1-0.5 mM) of tetracaine shifted V(gamma)BAR and partially suppressed the tetracaine-sensitive charge, resulting in a difference Q-V plot that rose to a peak an then decayed to a plateau level. Following a TEST pulse to > -60 mV, the slow inward current component during a post-pulse to approximately -60 mV was also tetracaine sensitive. Tle 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 function 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 voltage-dependent components separated by all four methods are consistent with each other, and are therefore concluded to be equivalent to the same Q(gamma) component. The shortcomings of each separation method are critically discussed. Since each method has its own advantages and disadvantages, it is recommended that, as much as possible, Q(gamma) should be separated by more than one method to obtain more reliable results.