Charge Movement in Cut Twitch Fibres of Rana Temporaries Containing 0.1 mm EGTA

Abstract

Charge movement was studied in the cut twitch fibres of Rana temporaria with a double Vaseline‐gap voltage‐clamp technique. In fibres containing 0.1 mm EGTA, the Iγ hump component of charge movement was either unresolvable or had a brief duration at all potentials. In the steady‐state charge‐voltage (Q–V) plots that were separable into the less voltage dependent (Qβ) and more voltage dependent (Qγ) components, the amount of Qγ was 0.9 ± 0.1 nC μF−1 (mean ±s.e.m., n= 18). Fibres containing 20 mm EGTA showed broad Iγ humps at about −45 mV. The addition of 1.8 mm total Ca2+ to the end‐pools accelerated the rising phase and abbreviated the duration of the hump. I γ humps in fibres containing 0.1 mm EGTA resembled those in intact fibres when recorded at similar low temperatures. These results explain the controversy concerning the variable appearance of Iγ humps in cut fibres.

A slow calcium‐dependent component of charge movement in Rana temporaria cut twitch fibres

“…The internal solution contained 0.1 mM EGTA. The I γ humps in the ON transients had very fast kinetics, consistent with the finding of Hui & Chen (1997). This made it difficult to separate visually the I γ hump from the I β component.…”

“…When the internal solution contains 20 mM EGTA without added Ca 2+ , I γ has a much delayed time course with respect to I β in the ON segment of charge movement traces, and I δ is absent in both ON and OFF segments. When the internal solution contains 0.1 mM EGTA or 20 mM EGTA plus 1.8 mM Ca 2+ , I γ is accelerated such that it has a time course comparable to that of I β in the ON segment of charge movement traces, and the amount of Q γ is reduced (Hui & Chen, 1997). Moreover, a slow I δ is present in both ON and OFF segments and the amount of Q δ is surprisingly large (see below).…”

“…When the myoplasmic Ca 2+ in cut fibres is chelated by 20 mM EGTA, the ON I γ component is manifested as a prominent hump and Q γ accounts for about half of the total charge (Hui & Chandler, 1990; Hui, 1991 a ). When the [Ca 2+ ] i is restored to the physiological level, the amount of Q γ is very much reduced and the remaining I γ is accelerated (Hui & Chen, 1997). In this paper, results are presented to show that although I γ is reduced in the latter situation, slow I δ appears in both the ON and OFF segments of TEST ‐ CONTROL current traces.…”

“…In measuring charge movement in cut fibres, we routinely used 20 mM EGTA in the end‐pool solution to suppress fibre contraction (beginning with Hui & Chandler, 1990). The presence of EGTA has the additional advantages of stabilizing the fibre for several hours, suppressing the calcium‐dependent Cl − current (Hui & Chen, 1994) and enhancing the appearance of the I γ hump (Hui & Chen, 1997). However, this high [EGTA] i reduces the resting free [Ca 2+ ] in the myoplasm to far below the physiological level, thereby reducing the Ca 2+ reloading of the sarcoplasmic reticulum (SR) after activities.…”

Effects of ryanodine on calcium sparks in cut twitch fibres of Rana temporaria

“…An alert of a progressive change in the physiological state of the cut fibre preparation over time was issued by Maylie et al (1987). Moreover, the 0.1 mM [EGTA] i used in the internal solution is not as ideal as a high concentration, such as 20 mM, in stabilizing and maintaining the health of cut fibres, as remarked by Hui & Chen (1997).…”