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4. The reversal potential for the early current appears to be consistent with the sodium equilibrium potential expected in hypertonic solutions.5. The variation of the equilibrium potential for the delayed current (VE) with external potassium concentration suggests that the channel for delayed current has a ratio of potassium to sodium permeability of 30:1; this is less than the resting membrane where the ratio appears to be 100: 1. VE corresponds well with the membrane potential at the beginning of the negative after-potential observed under similar conditions. 6. The variation of VE with the amount of current which has passed through the delayed channel suggests that potassium ions accumulate in a space of between X and W of the fibre volume. If potassium accumulates in the transverse tubular system (T system) much greater variation in VE would be expected.

SUMMARY1. Single twitch fibres, dissected from frog muscle, were injected with the metallochromic dye Arsenazo III. Changes in dye-related absorbance measured at 650 or 660 nm were used to estimate the time course of myoplasmic free [Ca2+] following either action potential stimulation or voltage-clamp depolarization (temperature, 15-17 0C).2. The amplitude of the Ca2+ transient decreased when fibres were stretched to sarcomere spacings approaching 4 /sm. The effect appeared to be less marked in H20 Ringer than in D20 Ringer, where a reduction of about 40 % was observed in going from 3 0 psm to 3 7-3 9 ,sm.3. In fibres heavily injected with dye (1-5-2-2 mM-dye) at least 0-1 mM-Ca2+ was complexed with Arsenazo III following a single action potential, implying that at least 041 mM-Ca2+ was released from the sarcoplasmic reticulum (s.r.) into the myoplasm.4. Computer simulations were carried out to estimate the flux of Ca2+ between the s.r. and myoplasm (in fibres containing no more that 0-8 mM-dye). The amounts and time courses of Ca2+ bound to the Ca2+-regulatory sites on troponin and to the Ca2+, Mg2+ sites on parvalbumin were estimated from the free [Ca2+] wave form and the law of mass action. In the computations the total myoplasmic [Ca2+] was taken as the total amount of Ca2+ existing either as free ion or as ion complexed with dye, troponin or parvalbumin. The time derivative of total myoplasmic [Ca2+] was used as an estimate of net Ca2+ flux (release minus uptake) from the s.r. into myoplasm. Rate constants for formation of cation: receptor complex were taken from published values. For the Ca2+-regulatory sites on troponin, three sets of rate constants, corresponding to two values of dissociation constant (0-2 and 2 /IM) were used. Each set of three simulations was carried out both with and without parvalbumin.5. The simulations show that following action potential stimulation, 0-2-0-3 mMCa2+ enters the myoplasm from the s.r. The wave form of s.r. Ca2+ release is early and brief compared with the wave form of free [Ca2+]. Neither the selection of troponin rate constants nor the inclusion ofparvalbumin has much effect on the shape 626 S. M. BA YLOR, W. K. CHANDLER AND M. W. MARSHALL of the release wave form; the main effect of varying these parameters is to change the magnitude.6. After the initial, rapid phase of Ca2+ release from the s.r. there is a longer, maintained period of Ca2+ uptake. The peak magnitude of this uptake divided by the concentration of s.r. Ca2+ pump sites is used to estimate the turnover rate of the sites. The numbers so obtained, 2-4-6-3 s-1, are in good agreement with previously published values based on biochemical experiments. Thus, the known biochemical properties of the s.r. Ca2+ pump can explain the final phase of the computed s.r. Ca2+ flux and therefore, within the framework of the model, the falling phase of the free [Ca2+] transient.7. Simulations, based on Ca2+ transients obtained during a train of action potentials spaced 10 ms apart, indicate that s.r. Ca2+ release associ...

Sarcoplasmic reticulum (SR) Ca release was studied at 13-16~ in cut fibers (sarcomere length, 3.4-3.9 I~m) mounted in a double Vaseline-gap chamber. The amplitude and duration of the action-potential stimulated free [Ca] transient were reduced by equilibration with end-pool solutions that contained 20 mM EGTA with 1.76 mM Ca and 0.63 mM phenol red, a maneuver that appeared to markedly reduce the amount of Ca complexed by troponin. A theoretical analysis shows that, under these conditions, the increase in myoplasmic free [Ca] is expected to be restricted to within a few hundred nanometers of the SR Ca release sites and to have a time course that essentially matches that of release. Furthermore, almost all of the Ca that is released from the SR is expected to be rapidly bound by EGTA and exchanged for protons with a 1:2 stoichiometry. Consequently, the time course of SR Ca release can be estimated by scaling the ApH signal measured with phenol red by -13/2. The value of 13, the buffering power of myoplasm, was determined in fibers equilibrated with a combination of EGTA, phenol red, and fura-2; its mean value was 22 mM/pH unit. The Ca content of the SR (expressed as myoplasmic concentration) was estimated from the total amount of Ca released by either a train of action potentials or a depleting voltage step; its mean value was 2,685 I~M in the action-potential experiments and 2,544 IxM in the voltage-clamp experiments. An action potential released, on average, 0.14 of the SR Ca content with a peak rate of release of,'~5 %/ms. A second action potential, elicited 20 ms later, released only 0.6 times as much Ca (expressed as a fraction of the SR content), probably because Ca inactivation of Ca release was produced by the first action potential. During a depolarizing voltage step to 60 mV, the rate of Ca release rapidly increased to a peak value of~3 %/ms and then decreased to a quasi-steady level that was only 0.6 times as large; this decrease was also probably due to Ca inactivation of Ca release. SR Ca release was studied with small step depolarizations that open no more than one SR Ca channel in 7

SUMMARY1. The kinetics of mechanical activation were examined in muscle fibres of the frog's sartorius muscle, using a voltage clamp to control membrane potential, tetrodotoxin to eliminate electrical activity and microscopic observations to determine the mechanical threshold.2. The strength-duration curve was determined over a range of membrane potentials varying between -52 mV (rheobase) and + 90 mV. At 40 C the critical duration was about 11 msec at -30 mV, 4 msec at 0 mV and 2 msec at + 40 mV.3. For pulses where V > -10 mV the threshold criterion at 40 C was that the 'area above -30 mV' must exceed about 120 mV msec.4. The effect of a brief subthreshold pulse declines with a time constant of about 3 msec at -100 mV and about 8 msec at -85 mV at 40 C.5. Although the strength-duration curve is well fitted by assuming a first-order mechanism in which the rate of release of activator increases with membrane potential, other experiments show that the over-all mechanism is probably second order in time.6. A short pulse must be at least 50 % threshold if it is to give a visible contraction when added to a long pulse which is just below rheobase.7. Delayed rectification was conspicuous with medium or long pulses which were just below the mechanical threshold, but short pulses could give contraction without turning on any appreciable potassium conductance.8. The Appendix extends Falk's (1968) treatment of the charging of the tubular system under a voltage clamp.