A slow component of intramembranous charge movement during sarcoplasmic reticulum calcium release in frog cut muscle fibers

Cut muscle fibers from Rana temporaria were mounted in a double Vaseline-gap chamber and equilibrated with an end-pool solution that contained 20 mM EGTA and 1.76 mM Ca (sarcomere length, 3.3-3.8 microns; temperature, 14-16 degrees C). Sarcoplasmic reticulum (SR) Ca release, delta[CaT], was estimated from changes in myoplasmic pH (Pape, P.C., D.- S. Jong, and W.K. Chandler. 1995. J. Gen. Physiol. 106:259-336). The maximal value of delta[CaT] obtained during a depleting depolarization was assumed to equal the SR Ca content before stimulation, [CaSR]R (expressed as myoplasmic concentration). After a depolarization to -55 to -40 mV in fibers with [CaSR]R = 1,000-3,000 microM, currents from intramembranous charge movement, Icm, showed an early I beta component. This was followed by an I gamma hump, which decayed within 50 ms to a small current that was maintained for as long as 500 ms. This slow current was probably a component of Icm because the amount of OFF charge, measured after depolarizations of different durations, increased according to the amount of ON charge. Icm was also measured after the SR had been depleted of most of its Ca, either by a depleting conditioning depolarization or by Ca removal from the end pools followed by a series of depleting depolarizations. The early I beta component was essentially unchanged by Ca depletion, the I gamma hump was increased (for [CaSR]R > 200 microM), the slow component was eliminated, and the total amount of OFF charge was essentially unchanged. These results suggest that the slow component of ON Icm is not movement of a new species of charge but is probably movement of Q gamma that is slowed by SR Ca release or some associated event such as the accompanying increase in myoplasmic free [Ca] that is expected to occur near the Ca release sites. The peak value of the apparent rate constant associated with this current, 2-4%/ms at pulse potentials between -48 and -40 mV, is decreased by half when [CaSR]R approximately equal to 500-1,000 microM, which gives a peak rate of SR Ca release of approximately 5-10 microM/ms.     

A damped oscillation in the intramembranous charge movement and calcium release flux of frog skeletal muscle fibers

Asymmetric membrane currents and calcium transients were recorded simultaneously from cut segments of frog skeletal muscle fibers voltage clamped in a double Vaseline-gap chamber in the presence of high concentration of EGTA intracellularly. An inward phase of asymmetric currents following the hump component was observed in all fibers during the depolarization pulse to selected voltages (congruent to -45 mV). The average value of the peak inward current was 0.1 A/F (SEM = 0.01, n = 18), and the time at which it occurred was 34 ms (SEM = 1.8, n = 18). A second delayed outward phase of asymmetric current was observed after the inward phase, in those experiments in which hump component and inward phase were large. It peaked at more variable time (between 60 and 130 ms) with amplitude 0.02 A/F (SEM = 0.003, n = 11). The transmembrane voltage during a pulse, measured with a glass microelectrode, reached its steady value in less than 10 ms and showed no oscillations. The potential was steady at the time when the delayed component of asymmetric current occurred. ON and OFF charge transfers were equal for all pulse durations. The inward phase moved 1.4 nC/microF charge (SEM = 0.8, n = 6), or about one third of the final value of charge mobilized by these small pulses, and the second outward phase moved 0.7 nC/microF (SEM = 0.8, n = 6), bringing back about half of the charge moved during the inward phase. When repolarization intersected the peak of the inward phase, the OFF charge transfer was independent of the repolarization voltage in the range -60 to -90 mV. When both pre- and post-pulse voltages were changed between -120 mV and -60 mV, the equality of ON and OFF transfers of charge persisted, although they changed from 113 to 81% of their value at -90 mV. The three delayed phases in asymmetric current were also observed in experiments in which the extracellular solution contained Cd2+, La3+ and no Ca2+. Large increases in intracellular [Cl-] were imposed, and had no major effect on the delayed components of the asymmetric current. The Ca2+ transients measured optically and the calculated Ca2+ release fluxes had three phases whenever a visible outward phase followed the inward phase in the asymmetric current. Several interventions intended to interfere with Ca release, reduced or eliminated the three delayed phases of the asymmetric current.(ABSTRACT TRUNCATED AT 400 WORDS)     

Q beta and Q gamma components of intramembranous charge movement in frog cut twitch fibers

Intramembranous charge movement was measured in frog cut twitch fibers mounted in a double Vaseline-gap chamber with a TEA.Cl solution at 13-14 degrees C in the central pool. When a fiber was depolarized from a holding potential of -90 mV to a potential near -60 mV, the current from intramembranous charge movement was outward in direction and had an early, rapid component and a late, more slowly developing component, referred to as I beta and I gamma, respectively (1979. J. Physiol. [Lond.]. 289:83-97). When the pulse to -60 mV was preceded by a 100-600-ms pulse to -40 mV, early I beta and late I gamma components were also observed, but in the inward direction. The shape of the Q gamma vs. voltage curve can be estimated with this two-pulse protocol. The first pulse to voltage V allows the amounts of Q beta and Q gamma charge in the active state to change from their respective resting levels, Q beta (-90) and Q gamma (-90), to new steady levels, Q beta (V) and Q gamma (V). A second 100-120-ms pulse, usually to -60 mV, allows the amount of Q beta charge in the active state to change from Q beta (V) to Q beta (-60) but is not sufficiently long for the amount of Q gamma charge to change completely from Q gamma (V) to Q gamma (-60). The difference between the amount of Q gamma charge at the end of the second pulse and Q gamma (-60) is estimated from the OFF charge that is observed on repolarization to -90 mV. The OFF charge vs. voltage data were fitted, with gap corrections, with a Boltzmann distribution function plus a constant. The mean values of V (the potential at which, in the steady state, charge is distributed equally between the resting and active states) and k (the voltage dependence factor) were -59.2 mV (SEM, 1.1 mV) and 1.2 mV (SEM, 0.6 mV), respectively. The one-pulse charge vs. voltage data from the same fibers were fitted with a sum of two Boltzmann functions (1990. J. Gen. Physiol. 96:257-297). The mean values of V and k for the steeply voltage-dependent Boltzmann function, which is likely to be associated with the Q gamma component of charge, were -55.3 mV (SEM, 1.3 mV) and 3.3 mV (SEM, 0.6 mV), respectively, similar to the corresponding values obtained with the two-pulse protocol.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reduction of calcium inactivation of sarcoplasmic reticulum calcium release by fura-2 in voltage-clamped cut twitch fibers from frog muscle

Cut fibers from Rana temporaria and Rana pipiens (striation spacing, 3.9-4.2 microns) were mounted in a double Vaseline-gap chamber and studied at 14 degrees C. The Ca indicator purpurate-3,3' diacetic acid (PDAA) was introduced into the end pools and allowed to diffuse into the optical recording site. When the concentration at the site exceeded 2 mM, step depolarizations to 10 mV were applied and the [Ca] transient measured with PDAA was used to estimate Ca release from the sarcoplasmic reticulum (SR) (Baylor, S. M., W. K. Chandler, and M. W. Marshall. 1983. Journal of Physiology. 344:625-666). With depolarization, the rate of SR Ca release increased to an early peak and then rapidly decreased several-fold to a quasi-steady level. The total amount of Ca released from the SR at the time of peak rate of release appeared to be independent of SR Ca content, consistent with the idea that a single activated channel might pass, on average, a fixed number of ions, independent of the magnitude of the single channel flux. A possible explanation of this property is given in terms of locally induced Ca inactivation of Ca release. The solution in the end pools was then changed to one with PDAA plus fura-2. SR Ca release was estimated from the [Ca] transient, as before, and from the delta [Cafura-2] signal. On average, 2-3 mM fura-2 increased the quasi-steady level of the rate of SR Ca release by factors of 6.6 and 3.8, respectively, in three fibers from Rana temporaria and three fibers from Rana pipiens. The peak rate of release was increased in five of the six fibers but to a lesser extent than the quasi-steady level. In all fibers, the amplitude of the free [Ca] transient was markedly reduced. These increases in the rate of SR Ca release are consistent with the idea that Ca inactivation of Ca release develops during a step depolarization to 10 mV and that 2-3 mM fura-2 is able to reduce this inactivation by complexing Ca and thereby reducing free [Ca]. Once the concentration of fura-2 becomes sufficiently large, a further increase reduces the rate of SR Ca release. On average, 5-6 mM fura-2 increased the quasi-steady rate of release, compared with 0 mM fura-2, by 6.5 and 2.9, respectively, in four fibers from Rana temporaria and three from Rana pipiens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Measuring real-time sarcoplasmic reticulum calcium pumping in skinned muscle fibers

A method and a device for direct measurements of accumulation of calcium in the sarcoplasmic reticulum (SR) and its release from SR as a function of free Ca2+ in bath have been developed. About 30% of the volume inside muscle fibers of swimbladder of Opsanus tau is occupied by SR. A set of solutions was prepared for fiber dissection and making holes in outer membrane without destruction of membranes of the sarcoplasmic reticulum. Calcium was unloaded from SR using EGTA as a pCa buffer. Then solutions with 50-100 microM CaFURA2 or CabisFURA2 were used as pCa-buffer and fluorescent Ca-indicators for measurement of Ca exchange between a fiber with a volume of approximately 10 nl and a solution in the cuvette with a volume of 5 microl. An increase in fluorescence signified an increase in unbound FURA in the bath since Ca2+ pumped into the SR was removed from the bath. The slope represented the rate of Ca2+ uptake by the SR in the muscle fiber, the maximum being about 1.6 M/s per liter of solution in bath or 2.6 mM/s per liter of SR volume. In solutions without oxalate and Ruthenium Red, more Ca2+ was taken up by the SR, and oscillations of the bath free FURA level were often observed, which can be explained by calcium-induced calcium release.     

Pharmacological dissection of charge movement in frog skeletal muscle fibers.

When charge movement is measured from muscle fibers bathed in a moderately hypertonic solution, a secondary hump appears in the decay phase of the signal during the "on" of the test pulse. The hump can be suppressed by the application of dantrolene sodium or tetracaine. The amount of charge associated with the hump is approximately 20-25% of the total charge. All the observed properties of the hump charge are consistent with the possibility that it is more closely associated with calcium release from the sarcoplasmic reticulum, and thus more relevant to excitation-contraction coupling, than the rest of the charge.     

Effect of fura-2 on action potential-stimulated calcium release in cut twitch fibers from frog muscle

Cut fibers (striation spacing, 3.6-4.2 microns) were mounted in a double Vaseline-gap chamber and studied at 14-15 degrees C. One or both of the Ca indicators fura-2 and purpurate-3,3' diacetic acid (PDAA) were introduced into the optical recording site by diffusion from the end pools. Sarcoplasmic reticulum (SR) Ca release was elicited by action potential stimulation. With resting [fura-2] = 0 mM at the optical site, the [Ca] transient measured with PDAA was used to estimate SR Ca release (Baylor, S.M., W.K. Chandler, and M.W. Marshall. 1983. Journal of Physiology. 344:625-666). With resting [fura-2] > 0 mM, the contribution from Ca complexation by fura-2 was added to the estimate. When resting [fura-2] was increased from 0 to 0.5-2 mM, both the amount of SR Ca release and the maximal rate of release were increased by approximately 20%. These results are qualitatively similar to those obtained in intact fibers (Baylor, S.M., and S. Hollingworth. 1988. Journal of Physiology. 403:151-192; Hollingworth, S., A. B. Harkins, N. Kurebayashi, M. Konishi, and S. M. Baylor. 1992. Biophysical Journal. 63:224-234) and are consistent with a reduction of Ca inactivation of SR Ca release produced by 0.5-2 mM fura-2. With resting [fura-2] > or = 2 mM, the PDAA [Ca] transient was reduced to nearly zero and SR Ca release could be estimated from delta [Cafura-2] alone. When resting [fura-2] was increased from 2-4 to 5-6 mM, both the amount of SR Ca release and the maximal rate of release were decreased by approximately half, consistent with a possible reduction of Ca- induced Ca release (Jacquemond, V., L. Csernoch, M. G. Klein, and M. F. Schneider. 1991. Biophysical Journal. 60:867-873) or a possible pharmacological effect of fura-2.     

Simulation of calcium sparks in cut skeletal muscle fibers of the frog

Spark mass, the volume integral of Delta F/F, was investigated theoretically and with simulations. These studies show that the amount of Ca2+ bound to fluo-3 is proportional to mass times the total concentration of fluo-3 ([fluo-3T]); the proportionality constant depends on resting Ca2+ concentration ([Ca2+]R). In the simulation of a Ca2+ spark in an intact frog fiber with [fluo-3T] = 100 microM, fluo-3 captures approximately one-fourth of the Ca2+ released from the sarcoplasmic reticulum (SR). Since mass in cut fibers is several times that in intact fibers, both with similar values of [fluo-3T] and [Ca2+]R, it seems likely that SR Ca2+ release is larger in cut fiber sparks or that fluo-3 is able to capture a larger fraction of the released Ca2+ in cut fibers, perhaps because of reduced intrinsic Ca2+ buffering. Computer simulations were used to identify these and other factors that may underlie the differences in mass and other properties of sparks in intact and cut fibers. Our spark model, which successfully simulates calcium sparks in intact fibers, was modified to reflect the conditions of cut fiber measurements. The results show that, if the protein Ca2+-buffering power of myoplasm is the same as that in intact fibers, the Ca2+ source flux underlying a spark in cut fibers is 5-10 times that in intact fibers. Smaller source fluxes are required for less buffer. In the extreme case in which Ca2+ binding to troponin is zero, the source flux needs to be 3-5 times that in intact fibers. An increased Ca2+ source flux could arise from an increase in Ca2+ flux through one ryanodine receptor (RYR) or an increase in the number of active RYRs per spark, or both. These results indicate that the gating of RYRs, or their apparent single channel Ca2+ flux, is different in frog cut fibers--and, perhaps, in other disrupted preparations--than in intact fibers.     

Calcium-gated calcium channels in sarcoplasmic reticulum of rabbit skinned skeletal muscle fibers

The action of ruthenium red (RR) on Ca2+ loading by and Ca2+ release from the sarcoplasmic reticulum (SR) of chemically skinned skeletal muscle fibers of the rabbit was investigated. Ca2+ loading, in the presence of the precipitating anion pyrophosphate, was monitored by a light-scattering method. Ca2+ release was indirectly measured by following tension development evoked by caffeine. Stimulation of the Ca2+ loading rate by 5 microM RR was dependent on free Ca2+, being maximal at pCa 5.56. Isometric force development induced by 5 mM caffeine was reversibly antagonized by RR. IC50 for the rate of tension rise was 0.5 microM; that for the extent of tension was 4 microM. RR slightly shifted the steady state isometric force/pCa curve toward lower pCa values. At 5 microM RR, the pCa required for half-maximal force was 0.2 log units lower than that of the control, and maximal force was depressed by approximately 16%. These results suggest that RR inhibited Ca2+ release from the SR and stimulated Ca2+ loading into the SR by closing Ca2+-gated Ca2+ channels. Previous studies on isolated SR have indicated the selective presence of such channels in junctional terminal cisternae.     

Intramembranous charge movement in frog cut twitch fibers mounted in a double vaseline-gap chamber

Intramembranous charge movement was measured in cut twitch fibers mounted in a double Vaseline-gap chamber with either a tetraethylammonium chloride (TEA.Cl) or a TEA2.SO4 solution (13-14 degrees C) in the central pool. Charge vs. voltage data were fitted by a single two-state Boltzmann distribution function. The average values of V (the voltage at which steady-state charge is equally distributed between the two Boltzmann states), k (the voltage dependence factor), and qmax/cm (the maximum charge divided by the linear capacitance, both per unit length of fiber) were V = -53.3 mV (SEM, 1.1 mV), k = 6.3 mV (SEM, 0.3 mV), qmax/cm = 18.0 nC/microF (SEM, 1.1 nC/microF) in the TEA.Cl solution; and V = -35.1 mV (SEM, 1.8 mV), k = 10.5 mV (SEM, 0.9 mV), qmax/cm = 36.3 nC/microF (SEM, 3.2 nC/microF) in the TEA2.SO4 solution. These values of k are smaller than those previously reported for cut twitch fibers and are as small as those reported for intact fibers. If a correction is made for the contributions of currents from under the Vaseline seals, V = -51.2 mV (SEM, 1.1 mV), k = 7.2 mV (SEM, 0.4 mV), qmax/cm = 22.9 nC/microF (SEM, 1.4 nC/microF) in the TEA.Cl solution; and V = -34.0 mV (SEM, 1.9 mV), k = 10.1 mV (SEM, 1.1 mV), qmax/cm = 38.8 nC/microF (SEM, 3.2 nC/microF) in the TEA2.SO4 solution. With this correction, however, the fit of the theoretical curve to the data is poor. A good fit with this correction can be obtained with a sum of two Boltzmann distribution functions. The first has average values V = -33.0 mV (SEM, 2.8 mV), k = 11.0 mV (SEM, 0.5 mV), qmax/cm = 10.6 nC/microF (SEM, 1.0 nC/microF) in the TEA.Cl solution; and V = -20.0 mV (SEM, 3.3 mV), k = 17.0 mV (SEM, 2.0 mV), qmax/cm = 36.4 nC/microF (SEM, 2.3 nC/microF) in the TEA2.SO4 solution. The second has average values V = -56.5 mV (SEM, 1.3 mV), k = 2.9 mV (SEM, 0.4 mV), qmax/cm = 13.2 nC/microF (SEM, 1.0 nC/microF) in the TEA.Cl solution; and V = -41.6 mV (SEM, 1.4 mV), k = 2.5 mV (SEM, 0.8 mV), qmax/cm = 11.8 nC/microF (SEM, 1.7 nC/microF) in the TEA2.SO4 solution. When a fiber is depolarized to near V of the second Boltzmann function, a slowly developing "hump" appears in the ON-segment of the current record.(ABSTRACT TRUNCATED AT 400 WORDS)     

The concentration of free Ca(2+) in the sarcoplasmic reticulum of frog cut twitch skeletal muscle fibers estimated with tetramethylmurexide

One aim of this article was to determine the resting concentration of free Ca²⁺ in the sarcoplasmic reticulum (SR) of frog cut skeletal muscle fibers ([Ca²⁺]_SR,R) using the calcium absorbance indicator dye tetramethylmurexide (TMX). Another was to determine the ratio of [Ca²⁺]_SR,R to TMX's apparent dissociation constant for Ca²⁺ (K_app) in order to establish the capability of monitoring [Ca²⁺]_SR(t) during SR Ca²⁺ release – a signal needed to determine the Ca²⁺ permeability of the SR. To reveal the properties of TMX in the SR, the surface membrane was rapidly permeabilized with saponin to rapidly dissipate myoplasmic TMX. Results indicated that the concentration of Ca-free TMX in the SR was 2.8-fold greater than that in the myoplasm apparently due to binding of TMX to sites in the SR. Taking into account that such binding might influence K_app as well as a dependence of K_app on TMX concentration, the results indicate an average [Ca²⁺]_SR,R ranging from 0.43 to 1.70 mM. The ratio [Ca²⁺]_SR,R/K_app averaged 0.256, a relatively low value which should not depend on factors influencing K_app. As a result, the time course of [Ca²⁺]_SR(t) in response to electrical stimulation is well determined by, and approximately linearly related to, the active TMX absorbance signal.     

Factors affecting the appearance of the hump charge movement component in frog cut twitch fibers.

Charge movement was measured in frog cut twitch fibers with the double Vaseline gap technique. Five manipulations listed below were applied to investigate their effects on the hump component (I gamma) in the ON segments of TEST minus CONTROL current traces. When external Cl-1 was replaced by MeSO3- to eliminate Cl current, I gamma peaked earlier due to a few millivolts shift of the voltage dependence of I gamma kinetics in the negative direction. The Q-V plots in the TEA.Cl and TEA.MeSO3 solutions were well fitted by a sum of two Boltzmann distribution functions. The more steeply voltage-dependent component (Q gamma) had a V approximately 6 mV more negative in the TEA.MeSO3 solution than in the TEA.Cl solution. These voltage shifts were partially reversible. When creatine phosphate in the end pool solution was removed, the I gamma hump disappeared slowly over the course of 20-30 min, partly due to a suppression of Q gamma. The hump reappeared when creatine phosphate was restored. When 0.2-1.0 mM Cd2+ was added to the center pool solution to block inward Ca current, the I gamma hump became less prominent due to a prolongation in the time course of I gamma but not to a suppression of Q gamma. When the holding potential was changed from -90 to -120 mV, the amplitude of I beta was increased, thereby obscuring the I gamma hump. Finally, when a cut fiber was stimulated repetitively, I gamma lost its hump appearance because its time course was prolonged. In an extreme case, a 5-min resting interval was insufficient for a complete recovery of the waveform. In general, a stimulation rate of once per minute had a negligible effect on the shape of I gamma. Of the five manipulations, MeSO3- has the least perturbation on the appearance of I gamma and is potentially a better substitute for Cl- than SO2-(4) in eliminating Cl current if the appearance of the I gamma hump is to be preserved.     

Real-time measurement of the rate of sarcoplasmic reticulum calcium pumping in skinned muscle fibers

A method and a device had been developed to directly measure the accumulation of calcium in the sarcoplasmic reticulum and its release from the sarcoplasmic reticulum, depending on the free Ca²⁺ concentration in the solution. The sarcoplasmic reticulum occupies to 30% of the volume of the swim bladder muscles of the oyster toadfish Opsanus tau. To isolate and skin muscle fibers and to remove the accumulated calcium from the sarcoplasmic reticulum, a set of solutions containing EGTA as a pCa buffer was used. To measure the calcium exchange between a fiber ～10 nl in volume and the solution in a 5-μl cuvette, instead of EGTA, 50–100 μM FURA2 or bisFURA2 was used both as pCa buffer and as a fluorescent indicator of the calcium concentration in the cuvette. An increase in fluorescence intensity meant an increase in the free FURA concentration in the solution surrounding the fiber since the calcium entering the sarcoplasmic reticulum was taken from this solution. The slope of the fluorescence curve corresponded to a rate of calcium accumulation in the sarcoplasmic reticulum of 1.6 μmol per second per liter of the solution in the cuvette or 2.6 mmol per second per liter of the sarcoplasmic reticulum. A solution without oxalate and ruthenium red may exhibit oscillations of the free FURA concentration, which can be explained by calcium-activated calcium release from the sarcoplasmic reticulum.     

Surface charge and calcium binding in sarcoplasmic reticulum membranes

Vesicles of fragmented sarcoplasmic reticulum membranes have been adsorbed on to 2.68 latex spheres. Observation of these vesicle containing spheres in the presence of an electric field allows a calculation of the electrophoretic mobility of the vesicles Following this determination, the net membrane surface charge has been estimated. The mobility of sarcoplasmic reticulum membranes exhibited a dependency on pH. At an ionic strength of 0.10 a mobility (pH=7.0) of –0.67±0.10/sec/volt/cm was observed. At pH=7.0 and /2=0.150 the net excess negative charge density was 2.0×10^–2 coul/m². This is equivalent to one charge per 10³ A² (assuming a uniform charge distribution). With an average vesicle volume of 2.8×10⁸ A³ and a surface area of 2×10⁶ A² the surface of one vesicle would contain a net of approximately 2×10³ negative charges. While the mobility did not change during uptake of calcium by the vesicles, both glutaraldehyde fixation and lecithin extraction by phospholipase C greatly altered the mobility of the vesicle membrane. Calcium binding and uptake both exhibited a dependence on pH.     

Kinetic properties of intramembrane charge movement under depolarized conditions in frog skeletal muscle fibers

Intramembrane charge movement was measured on skeletal muscle fibers of the frog in a single Vaseline-gap voltage clamp. Charge movements determined both under polarized conditions (holding potential, VH = -100 mV; Qmax = 30.4 +/- 4.7 nC/micro(F), V = -44.4 mV, k = 14.1 mV; charge 1) and in depolarized states (VH = 0 mV; Qmax = 50.0 +/- 6.7 nC/micro(F), V = -109.1 mV, k = 26.6 mV; charge 2) had properties as reported earlier. Linear capacitance (LC) of the polarized fibers was increased by 8.8 +/- 4.0% compared with that of the depolarized fibers. Using control pulses measured under depolarized conditions to calculate charge 1, a minor change in the voltage dependence (to V = -44.6 mV and k = 14.5 mV) and a small increase in the maximal charge (to Qmax = 31.4 +/- 5.5 nC/micro(F] were observed. While in most cases charge 1 transients seemed to decay with a single exponential time course, charge 2 currents showed a characteristic biexponential behavior at membrane potentials between -90 and -180 mV. The voltage dependence of the rate constant of the slower component was fitted with a simple constant field diffusion model (alpha m = 28.7 s-1, V = -124.0 mV, and k = 15.6 mV). The midpoint voltage (V) was similar to that obtained from the Q-V fit of charge 2, while the steepness factor (k) resembled that of charge 1. This slow component could also be isolated using a stepped OFF protocol; that is, by hyperpolarizing the membrane to -190 mV for 200 ms and then coming back to 0 mV in two steps. The faster component was identified as an ionic current insensitive to 20 mM Co2+ but blocked by large hyperpolarizing pulses. These findings are consistent with the model implying that charge 1 and the slower component of charge 2 interconvert when the holding potential is changed. They also explain the difference previously found when comparing the steepness factors of the voltage dependence of charge 1 and charge 2.     

Effects of D-600 on intramembrane charge movement of polarized and depolarized frog muscle fibers

Intramembrane charge movement has been measured in frog cut skeletal muscle fibers using the triple vaseline gap voltage-clamp technique. Ionic currents were reduced using an external solution prepared with tetraethylammonium to block potassium currents, and O sodium + tetrodotoxin to abolish sodium currents. The internal solution contained 10 mM EGTA to prevent contractions. Both the internal and external solutions were prepared with impermeant anions. Linear capacitive currents were subtracted using the P-P/4 procedure, with the control pulses being subtracted either at very negative potentials, for the case of polarized fibers, or at positive potentials, for the case of depolarized fibers. In 63 polarized fibers dissected from Rana pipiens or Leptodactylus insularis frogs the following values were obtained for charge movement parameters: Qmax = 39 nC/microF, V = 36 mV, k = 18.5 mV. After depolarization we found that the total amount of movable charge was not appreciably reduced, while the voltage sensitivity was much changed. For 10 fibers, in which charge movement was measured at -100 and at 0 mV, Qmax changed from 46 to 41 nC/microF, while V changed from -41 to -103 mV and k changed from 20.5 to 30 mV. Thus membrane depolarization to 0 mV produces a shift of greater than 50 mV in the Q-V relationship and a decrease of the slope. Membrane depolarization to -20 and -30 mV, caused a smaller shift of the Q-V relationship. In normally polarized fibers addition of D-600 at concentrations of 50-100 microM, does not cause important changes in charge movement parameters. However, the drug appears to have a use-dependent effect after depolarization. Thus in depolarized fibers, total charge is reduced by approximately 20%. D-600 causes no further changes in the voltage sensitivity of charge movement in fibers depolarized to 0 mV, while in fibers depolarized to -20 and -30 mV it causes the same effects as that obtained with depolarization to 0 mV. These results are compatible with the idea that after depolarization charge 1 is transformed into charge 2. D-600 appears to favor the conversion of charge 1 into charge 2. Since D-600 also favors contractile inactivation, charge 2 could represent the state of the voltage sensor for excitation-contraction coupling in the inactivated state.     

Calcium dependence of inactivation of calcium release from the sarcoplasmic reticulum in skeletal muscle fibers

The steady-state calcium dependence of inactivation of calcium release from the sarcoplasmic reticulum was studied in voltage-clamped, cut segments of frog skeletal muscle fibers containing two calcium indicators, fura-2 and anti-pyrylazo III (AP III). Fura-2 fluorescence was used to monitor resting calcium and relatively small calcium transients during small depolarizations. AP III absorbance signals were used to monitor larger calcium transients during larger depolarizations. The rate of release (Rrel) of calcium from the sarcoplasmic reticulum was calculated from the calcium transients. The equilibrium calcium dependence of inactivation of calcium release was determined using 200-ms prepulses of various amplitudes to elevate [Ca2+] to various steady levels. Each prepulse was followed by a constant test pulse. The suppression of peak Rrel during the test pulse provided a measure of the extent of inactivation of release at the end of the prepulse. The [Ca2+] dependence of inactivation indicated that binding of more than one calcium ion was required to inactivate each release channel. Half-maximal inactivation was produced at a [Ca2+] of approximately 0.3 microM. Variation of the prepulse duration and amplitude showed that the suppression of peak release was consistent with calcium-dependent inactivation of calcium release but not with calcium depletion. The same calcium dependence of inactivation was obtained using different amplitude test pulses to determine the degree of inactivation. Prepulses that produced near maximal inactivation of release during the following test pulse produced no suppression of intramembrane charge movement during the test pulse, indicating that inactivation occurred at a step beyond the voltage sensor for calcium release. Three alternative set of properties that were assumed for the rapidly equilibrating calcium-binding sites intrinsic to the fibers gave somewhat different Rrel records, but gave very similar calcium dependence of inactivation. Thus, equilibrium inactivation of calcium release appears to be produced by rather modest increases in [Ca2+] above the resting level and in a steeply calcium-dependent manner. However, the inactivation develops relatively slowly even during marked elevation of [Ca2+], indicating that a calcium-independent transition appears to occur after the initial calcium-binding step.