Calcium channel gamma subunits provide insights into the evolution of this gene family.

The gamma subunits of voltage-dependent calcium channels influence calcium current properties and may be involved in other physiological functions. Five distinct gamma subunits have been described from human and/or mouse. The first identified member of this group of proteins, gamma(1), is a component of the L-type calcium channel expressed in skeletal muscle. A second member, gamma(2), identified from the stargazer mouse regulates the targeting of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors to the postsynaptic membrane. We report here the identification of three novel gamma subunits from rat and mouse as well as the unidentified rat, mouse and human orthologs of the previously described subunits. Phylogenetic analysis of the 24 mammalian gamma subunits suggests the following relationship ((((gamma(2), gamma(3)), (gamma(4), gamma(8))), (gamma(5), gamma(7))), (gamma(1), gamma(6))) that indicates that they evolved from a common ancestral gamma subunit via gene duplication. Our analysis reveals that the novel gamma subunit gamma(6) most closely resembles gamma(1) and shares with it the lack of a PSD-95/DLG/ZO-1 (PDZ)-binding motif that is characteristic of most other gamma subunits. Rat gamma subunit mRNAs are expressed in multiple tissues including brain, heart, lung, and testis. The expression of gamma(1) mRNA and the long isoform of gamma(6) mRNA is most robust in skeletal muscle, while gamma(6) is also highly expressed in cardiac muscle. Based on our analysis of the molecular evolution, primary structure, and tissue distribution of the gamma subunits, we propose that gamma(1) and gamma(6) may share common physiological functions distinct from the other homologous gamma subunits.

Elevated growth hormone increases the Ca2+ sensitivity of slow- and fast-twitch skeletal muscle of female rats.

To determine the effect of plasma growth hormone (GH) on skeletal muscle function, we measured the free Ca2+ concentration-tension relationship of slow-twitch (soleus) and fast-twitch (peroneus longus) muscles isolated from rats undergoing acromegaly in response to implanted, GH-secreting tumors. Muscles from adult (9 mo) and aged rats (24 mo) were studied after the tumor-bearing rats weighted over 50% more than their age-matched controls. Ca(2+)-activated isometric tension was recorded from skinned muscle fibers. For soleus muscles, the free Ca2+ concentration producing 50% of maximal tension ([Ca2+]50) was 2.0 microM for rats with tumors and 3.4-3.6 microM for controls. For peroneus longus fibers, [Ca2+]50 shifted from 6.1-6.7 microM in controls to 3.5 microM after tumors were introduced into either adult or aged rats. Soleus muscle fibers from neonatal rats (14 days) were less sensitive to Ca2+ than those isolated from adult rats, having a [Ca2+]50 of 7.3 microM. The Ca2+ sensitivity of peroneus longus fibers did not change with age. We conclude that significant increases in myofibrillar Ca2+ sensitivity occur in skeletal muscles undergoing rapid growth induced by GH-secreting tumors.

Effects of relaxin on rat atrial myocytes. I. Inhibition of I(to) via PKA-dependent phosphorylation.

The peptide hormone relaxin has direct, positive inotropic and chronotropic effects on rat hearts in vivo and in vitro. Relaxin's effects on the electrophysiological properties of single quiescent atrial cells from normal rats were investigated with a whole cell patch clamp. Relaxin had a significant inhibitory effect on outward potassium currents. The outward potassium current consisted of a transient component (I(to)) and a sustained component (I(S)). The addition of 100 ng/ml of relaxin inhibited the peak I(to) in a voltage-dependent manner (74% inhibition at a membrane potential of -10 mV to 30% inhibition at +70 mV). The time to reach peak I(to) and the apparent time constant of inactivation of I(to) were increased by relaxin. Dialysis with the protein kinase A inhibitor 5-24 amide (2 microM) prevented relaxin's effects, suggesting an obligatory role for this kinase in the relaxin-dependent regulation of the potassium current.

Effects of relaxin on rat atrial myocytes. II. Increased calcium influx derived from action potential prolongation.

Relaxin produces positive inotropic and chronotropic effects in rat hearts. The effect of relaxin on the action potential duration (APD) of single quiescent rat atrial cells was investigated with a whole cell patch clamp. Relaxin induced a significant, dose-dependent prolongation of the APD. This effect was maximal at 200 ng/ml (nominal concentration of 33.6 nM), which caused, on average, a 57% increase in the time taken to reach 90% repolarization. The effect of relaxin was blocked by the protein kinase A inhibitor 5-24 amide, indicating that its effect is mediated by an adenosine 3',5'-cyclic monophosphate-dependent mechanism. The increased APD induced by relaxin caused an enhanced entrance of calcium, with the charge carried through voltage-activated calcium channels increased by approximately 25%. This increase was not due to a direct modulation of calcium currents (20); rather, it was a consequence of the longer period of cellular depolarization. Our findings that relaxin increased the APD and therefore increased the calcium influx in atrial myocytes could explain the positive inotropic effects induced by relaxin in atrial preparations.

Antisense oligonucleotides against rat brain alpha1E DNA and its atrial homologue decrease T-type calcium current in atrial myocytes.

Low voltage-activated, or T-type, calcium currents are important regulators of neuronal and muscle excitability, secretion, and possibly cell growth and differentiation. The gene (or genes) coding for the pore-forming subunit of low voltage-activated channel proteins has not been unequivocally identified. We have used reverse transcription-PCR to identify partial clones from rat atrial myocytes that share high homology with a member of the E class of calcium channel genes. Antisense oligonucleotides targeting one of these partial clones (raE1) specifically block the increase in T-current density that normally results when atrial myocytes are treated with insulin-like growth factor 1 (IGF-1). Antisense oligonucleotides targeting portions of the neuronal rat alpha1E sequence, which are not part of the clones detected in atrial tissue, also block the IGF-1-induced increase in T-current, suggesting that the high homology to alpha1E seen in the partial clone may be present in the complete atrial sequence. The basal T-current expressed in these cells is also blocked by antisense oligonucleotides, which is consistent with the notion that IGF-1 up-regulates the same gene that encodes the basal current. These results support the hypothesis that a member of the E class of calcium channel genes encodes a low voltage-activated calcium channel in atrial myocytes.

Characterization of the potassium channel from frog skeletal muscle sarcoplasmic reticulum membrane.

1. The sarcoplasmic reticulum (SR) membrane of skeletal muscle contains potassium channels which are thought to support charge neutralization during calcium release by providing a permeability pathway for counter-ion movement. To describe the behaviour of the SR K+ channel under physiological conditions, single channel activity was recorded from excised patches of SR membrane. Patches were made from membrane blebs extruded from contracted muscle fibres whose surface membranes had been removed previously by mechanical dissection. 2. The channel was active over a large voltage range from -80 to +100 mV. The current-voltage relationship of the channel was linear over most of this voltage range (slope conductance equal to 60 pS in 130 mM potassium), but showed rectification at voltages below -50 mV. 3. The activity of the channel (number of state transitions per unit time) was greater at positive voltages than at negative voltages. Analysis of dwell-time distributions showed that the time spent in the open state is best fitted by a double Gaussian, suggesting that the channel possesses both a long (l)- and a short (s)-lived open state with identical conductances. The dwell times for the two states were Ts = 0.3 ms and Tl = 2.6 ms at +90 mV and Ts = 0.1 ms and Tl = 15.1 ms at -40 mV. Thus, positive voltage decreased the long open time significantly which was consistent with the observed increase in channel activity at positive potentials. 4. The permeability sequence of the channel to various monovalent cations was deduced from the channel reversal potential under bi-ionic conditions and was found to be: K+ > Rb+ > Na+ > Cs+ > Li+. 5. Channel activity was reduced when the patch was perfused with 1,10-bis-guanidino-n-decane (BisG10), a drug reported to block the SR K+ channel with high affinity. The drug concentration necessary to reduce the open probability (P(o)) by 50% was 19.8 microM at -40 mV and 338.2 microM at +50 mV. The zero voltage dissociation constant (Kd) was calculated to be 48 microM. 6. Pharmacological agents known to affect surface membrane K+ channels, such as 0.5 mM Ba2+ or 3.0 mM 4-aminopyridine, were much less effective in blocking the channel than BisG10. Physiological calcium concentrations (pCa = 8.0 and 3.0) did not affect channel behaviour.4

Inactivation of the sarcoplasmic reticulum calcium channel by protein kinase.

The ryanodine receptor protein of skeletal muscle sarcoplasmic reticulum (SR) membranes is a calcium ion channel which allows movement of calcium from the SR lumen into the cytoplasm during muscle activation. Gating of this channel is modulated by a number of physiologically important substances including calcium. Interestingly, calcium has both activating and inactivating effects which are concentration- and tissue-specific. In skeletal muscle, calcium-dependent inactivation of calcium release occurs at concentrations reached physiologically, suggesting that calcium may modulate the release process by a negative feedback mechanism. To determine the cellular mechanism responsible for calcium-dependent inactivation, we have investigated the ability of protein phosphorylation to affect single channel gating behaviour using the patch clamp technique. Here we demonstrate that the ryanodine receptor protein/calcium release channel of skeletal muscle SR is inactivated under conditions permissive for protein phosphorylation. This inactivation is reversed by the application of phosphatase and prevented by a peptide inhibitor specific for calcium/calmodulin-dependent protein kinase II. The results provide evidence for an endogenous protein kinase which is closely associated with the ryanodine receptor protein and regulates channel gating.

Postnatal changes in T-type calcium current density in rat atrial myocytes.

1. Postnatal changes in Ca2+ current were studied in voltage clamped atrial myocytes isolated from Sprague-Dawley rats. T- and L-type Ca2+ currents were identified using standard electrophysiological and pharmacological techniques. Cells were studied from seven groups of male and six groups of female rats ranging in age from 3 to 14 weeks. 2. The density of atrial T-type Ca2+ current showed significant variation during postnatal development, with a maximum density reached at 4.5-5 weeks. At this age, T-current density was 1.44 +/- 0.11 pA/pF (n = 23) for cells isolated from male and 1.25 +/- 0.09 pA/pF (n = 25) for cells isolated from female animals in bathing solutions containing 2 mM-Ca2+. T-current density in atrial cells isolated from younger animals (3.5 weeks postnatal) averaged 1.22 +/- 0.06 (n = 18) and 1.00 +/- 0.05 pA/pF (n = 22) or 85 and 80% of the maximum seen at 4.5-5 weeks for male and female rats, respectively. For rats older than 13 weeks, the average T-current density in atrial cells was 0.50 +/- 0.03 (n = 18) and 0.51 +/- 0.02 pA/pF (n = 35) or 35 and 41% of the maximum seen at 4.5-5 weeks for male and female rats, respectively. 3. In contrast to the T-type current, the density of atrial L-type Ca2+ current remained unchanged in rats from 3 to 14 weeks old. L-type current averaged 8.2 +/- 0.2 (n = 134) in male and 7.9 +/- 0.2 pA/pF (n = 102) in female rats. 4. Fluctuation analysis was used to estimate single T-channel current levels in 4.5- and 7.5-week-old male rats. While the T-current density differed by 70% at these two postnatal ages, no significant difference (P > 0.2) in single channel current was found. Single channel current was 0.12 +/- 0.01 pA (n = 9) for cells from 4.5-week-old and 0.13 +/- 0.01 pA (n = 7) for cells from 7.5-week-old rats. Currents were stimulated by test pulses from -80 to -30 mV at 5 mM-Ca2+. 5. No postnatal changes were seen in either the kinetics of activation or inactivation of macroscopic T-current.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium-induced inactivation of calcium release from the sarcoplasmic reticulum of skeletal muscle.

The ability of myofilament space Ca2+ to modulate Ca2+ release from the sarcoplasmic reticulum (SR) of skeletal muscle was investigated. Single fibers of the frog Rana pipiens belindieri were manually skinned (sarcolemma removed). Following a standard load and pre-incubation in varying myoplasmic Ca2+ concentrations, SR Ca2+ release was initiated by caffeine. Ca2+ release rates were calculated from the changes in absorbance of a Ca2+ sensitive dye, antipyrylazo III. An apparent dissociation constant (Kd) for dye-Ca2+ binding of 8000 microM 2 was determined by comparing the buffering action of the dye with that of ethylenebis(oxonitrilo)tetraacetate (EGTA) using the contractile proteins of the skinned fiber as a measure of free Ca2+. This value for Kd was used in the calculation of Ca2+ release rates. As the myoplasmic space Ca2+ was increased from pCa 7.4, Ca2+ release rates declined sharply such that at pCa 6.9 the calculated release rate was 72 +/- 3% (mean +/- SEM) of control (pCa 8.4). Further increases in myoplasmic Ca2+ from pCa 6.9 to pCa 6.1 did not result in a further decline in release rate. The effect of a decreased driving force on Ca2+ ions was investigated to determine whether it could account for the change in release rates observed. At pCa 6.9, where the greatest degree of inactivation occurred, the measured effects of a change in driving force could account for at most 40% of the observed inactivation. Varying concentrations of Ba2+ and Sr2+ in the myofilament space had no inactivating effect on the SR Ca2+ release rates. The ability of myofilament Ca2+ to inhibit SR Ca2+ release at concentrations normally encountered during muscle activation suggests a role for released Ca2+ as a modulator of the SR Ca2+ channel.

Decreased transient outward K+ current in ventricular myocytes from acromegalic rats.

Cardiac hypertrophy and heart failure are common to acromegalic patients who have abnormally high serum growth hormone (GH). While the function of cardiac muscle is clearly affected by chronically elevated GH, the electrical activity of myocytes from hearts with GH-dependent hypertrophy has not been studied. We used adult, female Wistar-Furth rats with induced GH-secreting tumors to study the effect of excessive GH on ion channels of cardiac myocytes. GH-secreting tumors were induced by subcutaneous inoculation of GH3 cells. Eight weeks after inoculation, the rats had doubled their body weight and heart size compared with age-matched controls. There were no differences in either action potential amplitude or resting potential of right ventricular myocytes from control and tumor-bearing rats. However, action potential duration increased significantly in tumor-bearing rats; the time to 50% repolarization was 23 +/- 14 ms (n = 10) compared with 6.6 +/- 1.5 ms (n = 14) in controls. The prolongation of the action potential was mainly due to a decrease in density of a transient outward current (It,o) carried by K+. The normalized conductance for It,o decreased from 0.53 +/- 0.10 nS/pF (n = 25) in controls to 0.33 +/- 0.09 nS/pF (n = 26) in tumor-bearing rats. The decrease in It,o) and increase in heart weight occurred with a similar time course. The increased action potential duration prolongs Ca2+ influx through L-type Ca2+ channels in the tumor-bearing animals; this may be important in cardiovascular adaptation.

The differentiation of excitability in embryonic chick limb motoneurons.

The well-documented role of neuromuscular activity as a regulator of motoneuron and muscle development raises important questions about the differentiation of excitability in motoneurons. We have recently described changes in expression of voltage-dependent calcium currents that take place during neuromuscular development in the chick embryo (McCobb et al., 1989). We now report similar analyses, using whole-cell patch-recording methods, of the major currents underlying action potential generation in the same motoneurons. Studies were conducted on identified hindlimb motoneurons isolated from the spinal cord at 3 very different stages of chick hindlimb development. Motoneurons could generate overshooting action potentials at the earliest stage studied [embryonic day 4 (E4)]. However, large changes in densities of several voltage-dependent ionic currents occurred thereafter. E6 and E11 motoneurons had progressively larger INa densities and, consequently, greater action potential amplitudes. Densities of 2 potassium currents, Ik and IA, increased on separate schedules. The relatively late and much larger increase in IA resulted in a substantial developmental decline in action potential duration. These changes, which will greatly affect motoneuron output to muscle by affecting Ca2+ entry through voltage-gated channels, occur at the same time that activity-dependent developmental changes occur in the neuromuscular system.

Increase in T-type calcium current in atrial myocytes from adult rats with growth hormone-secreting tumors.

Growth hormone (GH) has pronounced effects on protein synthesis and cell growth in cardiac muscle from adult animals, although the mechanism of its action is not understood. Because Ca2+ has been implicated as a regulator of mitogenic processes in a number of tissues, we investigated whether GH affects the transmembrane movement of Ca2+ through voltage-activated channels of cardiac myocytes. Atrial and ventricular myocytes were isolated from adult rats with GH-secreting tumors and studied electrophysiologically by using patch-clamp techniques. Tumor-bearing rats re-enter an active growth phase and double their body weight over age-matched controls 8 weeks after introduction of the tumor. Atrial myocytes from tumor-bearing animals showed a 3-fold increase in the density of T-type Ca2+ current compared with cells from control animals, although the voltage dependency of activation and inactivation of T-type current was not altered. The increase in T-current density of atrial myocytes preceded by at least a week any measurable change in heart weight, body weight, or myocyte size. L-type Ca2+ currents in atrial and ventricular cells were not affected. The results suggest that a tumor-derived growth factor, most likely GH, can cause a specific enhancement of T-type Ca2+ current in atrial myocytes.

Effect of perchlorate on calcium release in skinned fibres stimulated by ionic substitution and caffeine.

We have determined the effects of the perchlorate ion on the contracture of skinned (sarcolemma removed) skeletal muscle fibres, stimulated either by ionic substitution or caffeine. Calcium release was monitored in single cells by measuring the peak height of tension transients. Perchlorate significantly sensitizes fibres to activation by ionic substitution, a manipulation that is thought to trigger calcium release via the normal physiological pathway. Adding 0.8 mM perchlorate to the solutions shifted the curve relating the magnitude of ionic substitution to the level of activation leftward, such that smaller stimuli were needed to produce a contracture of a given height. Perchlorate could also trigger a contracture directly. Exposing fibres to 1.0 mM perchlorate caused contractures averaging 60% of bracketing controls. In contrast to contractures stimulated by ionic substitution, those triggered by caffeine were unaffected by perchlorate. Since caffeine is thought to act directly on the sarcoplasmic reticulum to cause calcium release, these results suggest that perchlorate enhances activation in skinned fibres by interacting with transverse tubular membranes.

Development alters the expression of calcium currents in chick limb motoneurons.

Calcium current types expressed in vertebrate spinal motoneurons have not been previously resolved. We have resolved three types in chick limb motoneurons identified by retrograde labeling and report that dramatic changes in their expression take place during development in vivo. T-, N-, and L-type calcium currents were distinguished on the basis of kinetics, voltage dependence, and unique pharmacological sensitivities. Developmental changes were characterized by studying motoneurons isolated from embryos at three stages spanning neuromuscular system development. T currents were dominant at the earliest stage. Motoneurons from embryos 2 days older showed much reduced T currents and much increased N and L currents. We suggest that mature motoneurons will be dominated by N- and L-type calcium currents and that T current may serve developmental roles.

Block of contracture in skinned frog skeletal muscle fibers by calcium antagonists.

The ability of a number of calcium antagonistic drugs including nitrendipine, D600, and D890 to block contractures in single skinned (sarcolemma removed) muscle fibers of the frog Rana pipiens has been characterized. Contractures were initiated by ionic substitution, which is thought to depolarize resealed transverse tubules in this preparation. Depolarization of the transverse tubules is the physiological trigger for the release of calcium ion from the sarcoplasmic reticulum and thus of contractile protein activation. Since the transverse tubular membrane potential cannot be measured in this preparation, tension development is used as a measure of activation. Once stimulated, fibers become inactivated and do not respond to a second stimulus unless allowed to recover or reprime (Fill and Best, 1988). Fibers exposed to calcium antagonists while fully inactivated do not recover from inactivation (became blocked or paralyzed). The extent of drug-induced block was quantified by comparing the height of individual contractures. Reprimed fibers were significantly less sensitive to block by both nitrendipine (10 degrees C) and D600 (10 and 22 degrees C) than were inactivated fibers. Addition of D600 to fibers recovering from inactivation stopped further recovery, confirming preferential interaction of the drug with the inactivated state. A concerted model that assumed coupled transitions of independent drug-binding sites from the reprimed to the inactivated state adequately described the data obtained from reprimed fibers. Photoreversal of drug action left fibers inactivated even though the drug was initially added to fibers in the reprimed state. This result is consistent with the prediction from the model. The estimated KI for D600 (at 10 degrees and 22 degrees C) and for D890 (at 10 degrees C) was approximately 10 microM. The estimated KI for nitrendipine paralysis of inactivated fibers at 10 degrees C was 16 nM. The sensitivity of reprimed fibers to paralysis by D600 and D890 was similar. However, inactivated fibers were significantly less sensitive to the membrane-impermeant derivative (D890) than to the permeant species (D600), which suggests a change in the drug-binding site or its environment during the inactivation process. The enantomeric dihydropyridines (+) and (-) 202-791, reported to be calcium channel agonists and antagonists, respectively, both caused paralysis, which suggests that blockade of a transverse tubular membrane calcium flux is not the mechanism responsible for antagonist-induced paralysis. The data support a model of excitation-contraction coupling involving transverse tubular proteins that bind calcium antagonists.

Physiological role and selectivity of the in situ potassium channel of the sarcoplasmic reticulum in skinned frog skeletal muscle fibers.

The role of K+ as a counterion during Ca2+ release from the sarcoplasmic reticulum (SR) has been investigated. An optical technique using the Ca2+-sensitive dye antipyrylazo III monitored Ca2+ release from skinned (sarcolemma removed) muscle fibers of the frog. Skinned fibers were used since the removal of the sarcolemma allows direct access to the SR membrane. Releases were stimulated by caffeine, which activates Ca2+ release directly by binding to a receptor on the SR. Two different methods were used to decrease the SR K+ conductance so that its effect on Ca2+ release could be assessed: (a) the SR K+ channel blocker, 1,10-bis-quanidino-n-decane (bisG10) was used to eliminate current pathways and (b) substitution of the impermeant ion choline for K+ was used to decrease charge carriers. Both bisG10 and choline substitution caused a concentration-dependent decrease in the Ca2+ release rate. Therefore we conclude that K+ is an important counterion for Ca2+ during its release from the SR. The selectivity of the in situ SR K+ channel to several monovalent cations was determined by substituting them for K+ and comparing their effect on Ca2+ release. The substituted ions were expected to affect Ca2+ release in proportion to their ability to support a counterion flux, which is, in turn, a function of their relative conductance through the SR K+ channel. The selectivity sequence determined by these experiments was K+ = Rb+ = Na+ greater than Cs+ greater than Li+ greater than choline.

Epileptic tendencies in relation to behavioral responses to a novel environment in the Mongolian gerbil.

The severity of epileptic-like seizures in gerbils (Meriones unguiculatus), placed in an open field, is directly related to their ambulatory activity on subsequent trials. An inverse relationship, however, occurs between seizure severity and oriented, bipedal rearing behavior on subsequent trials. Principal components and multiple linear regression analyses support the hypothesis that ambulation and rearing have different underlying neuronal mechanisms. If these two activities are considered as measures of arousal and attention, respectively, then epileptic-like seizures may be caused by hyperactivity of mechanisms which induce arousal.

Contractile activation and recovery in skinned frog muscle stimulated by ionic substitution.

Contractile activation of skinned (sarcolemma removed) skeletal muscle fibers stimulated by ionic substitution has been studied. Stimulating solutions contained varying amounts of Cl- and K+ with the [K+] x [Cl-] product kept constant at 368 mM2. Activation is a graded function of the ionic content of the stimulating solution. Mechanical threshold is reached when the [Cl-] is changed from 4 to 6.5 mM. Maximal activation occurs at 20 mM Cl-. After stimulation, fibers do not respond to a second stimulus unless allowed to recover. Contractile height reaches 50% of control levels after 30 s in the standard recovery solution (4 mM Cl). Full recovery is reached after approximately 2-4 min. The degree of contractile recovery after a constant interval (30 s) depends on the ionic composition of the recovery solution. Contractile recovery decreases 50% when the [Cl-] of the recovery solution is raised from 4 to 5.5 mM. The activation and recovery phenomena described for skinned fibers stimulated by ionic substitution are similar to those described for intact cells stimulated by elevated [K+]. This finding is consistent with the hypothesis that depolarization of resealed transverse tubules triggers release of Ca2+ from the sarcoplasmic reticulum of skinned fibers.

Calcium release rate in skinned skeletal muscle fibers measured with arsenazo III.

Calcium ion release from the sarcoplasmic reticulum of single skinned (sarcolemma removed) skeletal muscle fibers was studied using the calcium-sensitive dye arsenazo III (Arz III). Isotropic absorption measurements were made differentially to reduce the effect of movement artifacts. The question of dye stoichiometry was addressed by measuring the absorption ratio at 600 and 660 nm at various times during the calcium transient. The results indicate that little change in the proportions of the various calcium-dye species occurs until at least 1 s into the release and, further, that the 1:2 calcium-dye complex is unlikely to be the dominant species present at early times. The relationship between dye concentration and the slope of the early absorption change was found to be linear for all levels of fiber loading. This suggests that the 1:1 rather than the 2:2 complex is the major species formed at early times in skinned fibers, although this conclusion is at odds with in vitro studies of Arz III in solution. Beer's law was used to convert the slope of the absorption transient measured over the first 125 ms of a release to the rate of change of the calcium-dye complex. The average rate at which the calcium-dye complex was formed was found to be 0.6 microM/ms. Two models are considered that allow calculation of a correction factor that is used to convert this value to the rate of calcium release from the sarcoplasmic reticulum. The magnitude of these correction factors was a function of the dye and intrinsic buffer concentrations as well as the stoichiometry of the calcium-dye reaction. After application of the correction factors, the average release rate in our fibers was calculated to range from 0.8 to 13.5 microM/ms.

Potassium efflux from single skinned skeletal muscle fibers.

The efflux of 42K from single, skinned (sarcolemma removed) skeletal muscle fibers has been determined. Isotope washout curves are kinetically complex and can be fit as the sum of three exponentials, including a fast component (k = 0.25 s-1) with a pool size equivalent to 91% of the fiber volume, an intermediate component (k = 0.08 s-1) equivalent to 6% of the fiber volume, and a slow component (k = 0.008 s-1) equivalent to 0.5% of fiber volume. Only the intermediate kinetic component is significantly affected by pretreatment of fibers with detergent. Efflux curves from detergent-treated fibers could be fit as the sum of two exponentials with coefficients and rate constants comparable to those of the fast and slow component of washout of untreated controls. Similarly the washout of [14C]sucrose can be described as the sum of two exponentials. We conclude that the intermediate component of 42K washout results from the movement of ions from a membrane bound space within the skinned fiber. Because of its relative volume, the sarcoplasmic reticulum seems to be a reasonable choice as a structural correlate for this component. Our estimate of the potassium permeability for the sarcoplasmic reticulum (SR) based on the efflux data is 10(-7) cm/s. This value is less than previous estimates from isolated preparations.