Effects of membrane potential on the capacitance of skeletal muscle fibers

A method for measuring muscle fiber capacitance using small test pulses applied with the three-microelectrode voltage clamp is presented. Using this method, three membrane potential-dependent changes in capacitance were observed: (a) Capacitance of polarized fibers increased by 5--15% with depolarization from V less then -100 mV to voltages slightly below the contraction threshold. (b) Capacitance of fibers depolarized to -30 mV by 100 mM Rb solution decreased by roughly 8% with further depolarization to about +50 mV and increased with repolarization, exhibiting a maximum increase of about 10% at -80 to -90 mV. (c) Capacitance of fibers depolarized to -15 mV by 100 mM K solution increased by about 19% with further depolarization to +43 mV and decreased by about 23% with repolarization to -62 mV. Effects a and b are attributed to changes in specific membrane capacitance due to voltage-dependent redistribution of mobile charged groups within surface of T-tubule membranes. Effect c is caused by changes in the T-system space constant lambdaT due to the voltage dependence of K conductance (inward rectification). Analysis of c showed that in 100 mM K solution lambdaT congruent to 30 mum when inward rectification was fully activated by hyperpolarization and that the density of inward rectifier channels is about the same in surface and tubular membranes. Fiber internal resistance was found to be independent of voltage, a necessary condition for the interpretation of the capacitance measurements.     

Blockers of potassium current and resting membrane potential in rat muscle fibers.

Rat diaphragm fibers were equilibrated for several hours in 150 mM KCl; when they were returned to 5 mM KCl the resting potential went back to its original level with a half time of 17 min. This repolarization was blocked by 5 mM BaCl2, a blocker of the inward rectifier K channel. On the other hand, 0.1 mM apamin and 0.02 mM glibenclamide which block the Ca-dependent and ATP sensitive K channels, respectively, and 0.1 mM 9-AC a blocker of the Cl- channel did not affect the repolarization. 5 mM barium decreased the K conductance measured under current-clamp conditions in diaphragm muscle fibers. The possible role of the inward rectifier system in the repolarization following return to normal [K]o is discussed.     

The Effect of Glycerol Treatment of Voltage-Clamped Snake Muscle Fibers

The effect of glycerol treatment on the membrane currents and tension development was studied in voltage clamped snake muscle fibers. In muscle fibers which were exposed for 1 h to a normal saline containing 400 mM glycerol and then returned to a normal medium, graded depolarizations did not accompany contractile responses. However, when the fiber was depolarized to a certain level, an increment of outward current appeared which partially inactivated with time. The threshold for delayed rectification in glycerol-treated fibers was almost the same as that of intact fibers in spite of the absence of contractile tension. The results suggest that the delayed rectification may be attributed at least in part to the surface membrane and that the contractile activation probably does not depend simply on the inactivating outward currents through the delayed rectification channel.     

Voltage Clamp Experiments on Single Muscle Fibers of Rana pipiens

A voltage clamp for single muscle fibers has been developed. Stability of the system was achieved when an artificial node was created by enclosing a single muscle fiber in a petroleum jelly seal which served as an analogue of the myelin sheath. Typical voltage clamp records were obtained with large inward transient currents followed by a delayed rectification of the outward currents. These currents looked qualitatively similar when the transverse tubular system was destroyed. Errors in current measurement, especially those due to anomalous rectification, are discussed.     

Interaction between the cardiac rapidly (IKr) and slowly (IKs) activating delayed rectifier potassium channels revealed by low K+-induced hERG endocytic degradation

Cardiac repolarization is controlled by the rapidly (I(Kr)) and slowly (I(Ks)) activating delayed rectifier potassium channels. The human ether-a-go-go-related gene (hERG) encodes I(Kr), whereas KCNQ1 and KCNE1 together encode I(Ks). Decreases in I(Kr) or I(Ks) cause long QT syndrome (LQTS), a cardiac disorder with a high risk of sudden death. A reduction in extracellular K(+) concentration ([K(+)](o)) induces LQTS and selectively causes endocytic degradation of mature hERG channels from the plasma membrane. In the present study, we investigated whether I(Ks) compensates for the reduced I(Kr) under low K(+) conditions. Our data show that when hERG and KCNQ1 were expressed separately in human embryonic kidney (HEK) cells, exposure to 0 mM K(+) for 6 h completely eliminated the mature hERG channel expression but had no effect on KCNQ1. When hERG and KCNQ1 were co-expressed, KCNQ1 significantly delayed 0 mM K(+)-induced hERG reduction. Also, hERG degradation led to a significant reduction in KCNQ1 in 0 mM K(+) conditions. An interaction between hERG and KCNQ1 was identified in hERG+KCNQ1-expressing HEK cells. Furthermore, KCNQ1 preferentially co-immunoprecipitated with mature hERG channels that are localized in the plasma membrane. Biophysical and pharmacological analyses indicate that although hERG and KCNQ1 closely interact with each other, they form distinct hERG and KCNQ1 channels. These data extend our understanding of delayed rectifier potassium channel trafficking and regulation, as well as the pathology of LQTS.     

Tension relaxation after stretch in resting mammalian muscle fibers: stretch activation at physiological temperatures.

Tension responses to ramp stretches of 1-3% Lo (fiber length) in amplitude were examined in resting muscle fibers of the rat at temperatures ranging from 10 degrees C to 36 degrees C. Experiments were done using bundles of approximately 10 intact fibers isolated from the extensor digitorum longus (a fast muscle) and the soleus (a slow muscle). At low temperatures (below approximately 20 degrees C), the tension response consisted of an initial rise to a peak during the ramp followed by a complex tension decay to a plateau level; the tension decay occurred at approximately constant sarcomere length. The tension decay after a standard stretch at approximately 3-4.Lo/s contained a fast, an intermediate, and a (small amplitude) slow component, which at 10 degrees C (sarcomere length approximately 2.5 microns) were approximately 2000.s-1, approximately 150.s-1, and approximately 25.s-1 for fast fibers and approximately 2000.s-1, approximately 70.s-1 and approximately 8.s-1 for slow fibers, respectively. The fast component may represent the decay of interfilamentary viscous resistance, and the intermediate component may be due to viscoelasticity in the gap (titin, connectin) filament. The two- to threefold fast-slow muscle difference in the rate of passive tension relaxation (in the intermediate and the slow components) compares with previously reported differences in the speed of their active contractions; this suggests that "passive viscoelasticity" is appropriately matched to contraction speed in different muscle fiber types. At approximately 35 degrees C, the fast and intermediate components of tension relaxation were followed by a delayed tension rise at approximately 10.s-1 (fast fibers) and 2.5.s-1 (slow fibers); the delayed tension rise was accompanied by sarcomere shortening. BDM (5-10 mM) reduced the active twitch and tetanic tension responses and the delayed tension rise at 35 degrees C; the results indicate stretch sensitive activation in mammalian sarcomeres at physiological temperatures.     

A general procedure for determining the rate of calcium release from the sarcoplasmic reticulum in skeletal muscle fibers.

A general procedure for using myoplasmic calcium transients measured with a metallochromic indicator dye to calculate the time course of calcium release from the sarcoplasmic reticulum in voltage-clamped skeletal muscle fibers is described and analyzed. Explicit properties are first assigned to all relatively rapidly equilibrating calcium binding sites in the myoplasm so that the calcium content (CaF) in this pool of "fast" calcium can be calculated from the calcium transient. The overall properties of the transport systems and relatively slowly equilibrating binding sites that remove calcium from CaF are then characterized experimentally from the decay of CaF following fiber repolarization. The rate of calcium release can then be calculated as dCaF/dt plus the rate of removal of calcium from CaF. Two alternatives are assumed for the component of CaF that is due to fast binding sites intrinsic to the fiber: a linear instantaneous buffer or a set of binding sites having properties similar to thin filament troponin. Both assumptions yielded similar calcium release wave forms. Three alternative methods for characterizing the removal system are presented. The choice among these or other methods for characterizing removal can be based entirely on convenience since any method that reproduces the decay of CaF following fiber repolarization will give the same release wave form. The calculated release wave form will be accurate provided that the properties assumed for CaF are correct, that release turns off within a relatively short time after fiber repolarization, that the properties of the slow removal system are the same during and after fiber depolarization, and that possible spatial nonuniformities of free or bound calcium do not introduce major errors.     

Some electrical properties of the membrane of the barnacle muscle fibers under internal perfusion.

Intracellular perfusion technique has been applied to the muscle fibers of the barnacle species, Balanus nubilus. In these fibers, generation and the form of the calcium spike was governed by the frequency of stimulation and intra- and extracellular calcium concentrations. Voltage-clamp experiments showed that the magnitude of the potassium outward current was controlled by the intracellular calcium concentration whose increase, nearly 10(3)-fold, raised the resting membrane conductance and the outward potassium current. On the other hand, application of 10 mM zinc ions inside the muscle fiber had no effect on either the resting potential or the outward potassium current but suppressed the early inward calcium current. Similarly, the inward calcium current was decreased by low concentration of sodium ions in the extracellular fluid only when its ionic strength was made low by substituting sucrose for the sodium salt. Measurement of outward current with the muscle fiber in calcium-free ASW solution and intracellularly perfused with several cationic solutions established the selectivity sequence TEA less than Cs less than Li less than Tris less than Rb less than Na less than K for the potassium channel.     

[K+] dependence of open-channel conductance in cloned inward rectifier potassium channels (IRK1, Kir2.1).

Potassium conduction through unblocked inwardly rectifying (IRK1, Kir2.1) potassium channels was measured in inside-out-patches from Xenopus oocytes, after removal of polyamine-induced strong inward rectification. Unblocked IRK1 channel current-voltage (I-V) relations show very mild inward rectification in symmetrical solutions, are linearized in nonsymmetrical solutions that bring the K+ reversal potential to extreme negative values, and follow Goldman-Hodgkin-Katz constant field equation at extreme positive E alpha. When intracellular K+ concentration (KIN) was varied, at constant extracellular K+ concentration (KOUT) the conductance at the reversal potential (GREV) followed closely the predictions of the Goldman-Hodgkin-Katz constant field equation at low concentrations and saturated sharply at concentrations of > 150 mM. Similarly, when KOUT was varied, at constant KIN, GREV saturated at concentrations of > 150 mM. A square-root dependence of conductance on KOUT is a well-known property of inward rectifier potassium channels and is a property of the open channel. A nonsymmetrical two-site three-barrier model can qualitatively explain both the I-V relations and the [K+] dependence of conductance of open IRK1 (Kir2.1) channels.     

The delayed rectifier potassium conductance in the sarcolemma and the transverse tubular system membranes of mammalian skeletal muscle fibers

A two-microelectrode voltage clamp and optical measurements of membrane potential changes at the transverse tubular system (TTS) were used to characterize delayed rectifier K currents (IK(V)) in murine muscle fibers stained with the potentiometric dye di-8-ANEPPS. In intact fibers, IK(V) displays the canonical hallmarks of K(V) channels: voltage-dependent delayed activation and decay in time. The voltage dependence of the peak conductance (gK(V)) was only accounted for by double Boltzmann fits, suggesting at least two channel contributions to IK(V). Osmotically treated fibers showed significant disconnection of the TTS and displayed smaller IK(V), but with similar voltage dependence and time decays to intact fibers. This suggests that inactivation may be responsible for most of the decay in IK(V) records. A two-channel model that faithfully simulates IK(V) records in osmotically treated fibers comprises a low threshold and steeply voltage-dependent channel (channel A), which contributes ～31% of gK(V), and a more abundant high threshold channel (channel B), with shallower voltage dependence. Significant expression of the IK(V)1.4 and IK(V)3.4 channels was demonstrated by immunoblotting. Rectangular depolarizing pulses elicited step-like di-8-ANEPPS transients in intact fibers rendered electrically passive. In contrast, activation of IK(V) resulted in time- and voltage-dependent attenuations in optical transients that coincided in time with the peaks of IK(V) records. Normalized peak attenuations showed the same voltage dependence as peak IK(V) plots. A radial cable model including channels A and B and K diffusion in the TTS was used to simulate IK(V) and average TTS voltage changes. Model predictions and experimental data were compared to determine what fraction of gK(V) in the TTS accounted simultaneously for the electrical and optical data. Best predictions suggest that K(V) channels are approximately equally distributed in the sarcolemma and TTS membranes; under these conditions, >70% of IK(V) arises from the TTS.     

Effects of low myoplasmic Mg2+ on calcium binding by parvalbumin and calcium uptake by the sarcoplasmic reticulum in frog skeletal muscle.

The effects of low intracellular free Mg2+ on the myoplasmic calcium removal properties of skeletal muscle were studied in voltage-clamped frog skeletal muscle fibers by analyzing the changes in intracellular calcium and magnesium due to membrane depolarization under various conditions of internal free [Mg2+]. Batches of fibers were internally equilibrated with cut end solutions containing two calcium indicators, antipyrylazo III (AP III) and fura-2, and different concentrations of free Mg2+ (25 microM-1 mM) obtained by adding appropriate total amounts of ATP and magnesium to the solutions. Changes in AP III absorbance were used to monitor [Ca2+] and [Mg2+] transients, whereas fura-2 fluorescence was mostly used to monitor resting [Ca2+]. Shortly after applying an internal solution containing less than 60 microM free Mg2+ to the cut ends of depolarized fibers most of the fibers exhibited spontaneous repetitive movements, suggesting that free internal Mg2+ might affect the activity of the sarcoplasmic reticulum (SR) calcium channels at rest. The spontaneous contractions generally subsided. In polarized fibers the maximal amplitude of the calcium transient elicited by a depolarizing pulse was about the same whatever the internal [Mg2+], but its decay after the end of the pulse slower in low [Mg2+]. In low [Mg2+] (less than 0.14 mM), the mean rate constant of decay obtained from fitting a single exponential plus a constant to the decay of the calcium transients was approximately 30% of its value in the control fibers (1 mM internal [Mg2+]). A model characterizing the main calcium removal properties of a frog skeletal muscle fiber, including the SR pump and the Ca-Mg sites on parvalbumin, was fitted to the decay of the calcium transients. Results of the fits show that in low internal [Mg2+] the slowing of the decay of the calcium transient can be well predicted by both a decreased rate of SR calcium uptake and an expected decreased resting magnesium occupancy of parvalbumin leading to a reduced contribution of parvalbumin to the overall rate of calcium removal. These results are thus consistent with the known properties of parvalbumin as a Ca-Mg buffer and furthermore suggest that in an intact portion of a muscle fiber, the activity of the SR calcium pump can be affected by the level of free Mg2+.     

Slow components of potassium tail currents in rat skeletal muscle

The kinetics of potassium tail currents have been studied in the omohyoid muscle of the rat using the three-microelectrode voltage-clamp technique. The currents were elicited by a two-pulse protocol in which a conditioning pulse to open channels was followed by a test step to varying levels. The tail currents reversed at a single well-defined potential (VK). At hyperpolarized test potentials (-100 mV and below), tail currents were inward and exhibited two clearly distinguishable phases of decay, a fast tail with a time constant of 2-3 ms and a slow tail with a time constant of approximately 150 ms. At depolarized potentials (-60 mV and above), tail currents were outward and did not show two such easily separable phases of decay, although a slow kinetic component was present. The slow kinetic phase of outward tail currents appeared to be functionally distinct from the slow inward tail since the channels responsible for the latter did not allow significant outward current. Substitution of Rb for extracellular K abolished current through the anomalous (inward-going) rectifier and at the same time eliminated the slow inward tail, which suggests that the slow inward tail current flows through anomalous rectifier channels. The amplitude of the slow inward tail was increased and VK was shifted in the depolarizing direction by longer conditioning pulses. The shift in VK implies that during outward currents potassium accumulates in a restricted extracellular space, and it is suggested that this excess K causes the slow inward tail by increasing the inward current through the anomalous rectifier. By this hypothesis, the tail current slowly decays as K diffuses from the restricted space. Consistent with such a hypothesis, the decay of the slow inward tail was not strongly affected by changing temperature. It is concluded that a single delayed K channel is present in the omohyoid. Substitution of Rb for K has little effect on the magnitude or time course of outward current tails, but reduces the magnitude and slows the decay of the fast component of inward tails. Both effects are consistent with a mechanism proposed for squid giant axon (Swenson and Armstrong, 1981): that (a) the delayed potassium channel cannot close while Rb is inside it, and (b) that Rb remains in the channel longer than K.     

Characterization of ion channels on the surface membrane of adult rat skeletal muscle.

The channels present on the surface membrane of isolated rat flexor digitorum brevis muscle fibers were surveyed using the patch clamp technique. 85 out of 139 fibers had a novel channel which excluded the anions chloride, sulfate, and isethionate with a permeability ratio of chloride to sodium of less than 0.05. The selectivity sequence for cations was Na+ = K+ = Cs+ greater than Ca++ = Mg++ greater than N-Methyl-D-Glucamine. The channel remained closed for long periods, and had a large conductance of approximately 320 pS with several subconductance states at approximately 34 pS levels. Channel activity was not voltage dependent and the reversal potential for cations in muscle fibers of approximately 0 mV results in the channel's behaving as a physiological leakage conductance. Voltage activated potassium channels were present in 65 of the cell attached patches and had conductances of mostly 6, 12, and 25 pS. The voltage sensitivity of the potassium channels was consistent with that of the delayed rectifier current. Only three patches contained chloride channels. The scarcity of chloride channels despite the known high chloride conductance of skeletal muscle suggests that most of the chloride channels must be located in the transverse tubular system.     

Effects of barium and ouabain on electrogenesis in various sites of intact and detubulated skeletal muscle fibers of the frog <Emphasis Type="Italic">R. temporaria</Emphasis>

In loose patch clamp experiments on intact sartorius muscle fibers of the frog Rana temporaria, there have been two types of waveforms of extracellularly recorded action potentials (AP). Responses of the first type (T1AP) consisted of an initial positive phase with a subsequent phase of strong negativity, the latter only in a few cases followed by a weak positive phase. Responses of the second type (T2AP) always had an additional positive phase concluding their waveform. In the detubulated fibers, only T1AP were recorded. Application of Ba²⁺ (10 μM) to the muscle led to a significant increase in the amplitude of the third T2AP phase whereas the T1AP characteristics of both intact and detubulated muscle preparations remained unchanged. In some of the studied intact fibers, after Ba²⁺ or ouabain (50 μM) applied, the latest positive signal phase was replaced by a negative phase. The amplitude of this latest negative phase was increased markedly by highfrequency stimulation. Under the simultaneous action of ouabain and Ba²⁺, there was a summation of their effects. Our results can be tentatively explained by that the T-system of a muscle fiber produces electrical responses substantially differing in their pattern and barium sensitivity from those transmitted across cell membranes. These differences could be resultant from the activity of T-tubular inwardly rectifying potassium channels (K_ir) and that of Na,K-ATPase as they both provide absorption of excessive extracellular potassium.     

Inwardly rectifying K(+) channels in esophageal smooth muscle

The whole cell patch-clamp technique was used to investigate whether there were inwardly rectifying K(+) (K(ir)) channels in the longitudinal muscle of cat esophagus. Inward currents were observable on membrane hyperpolarization negative to the K(+) equilibrium potential (E(k)) in freshly isolated esophageal longitudinal muscle cells. The current-voltage relationship exhibited strong inward rectification with a reversal potential (E(rev)) of -76.5 mV. Elevation of external K(+) increased the inward current amplitude and positively shifted its E(rev) after the E(k), suggesting that potassium ions carry this current. External Ba(2+) and Cs(+) inhibited this inward current, with hyperpolarization remarkably increasing the inhibition. The IC(50) for Ba(2+) and Cs(+) at -60 mV was 2.9 and 1.6 mM, respectively. Furthermore, external Ba(2+) of 10 microM moderately depolarized the resting membrane potential of the longitudinal muscle cells by 6.3 mV while inhibiting the inward rectification. We conclude that K(ir) channels are present in the longitudinal muscle of cat esophagus, where they contribute to its resting membrane potential.     

Repolarization of striated muscle fibers by the antifatigue agent,K-Mg-aspartate

Resting membrane potentials of isolated frog sartorius muscles were measured under a variety of conditions using intracellular glass microelectrodes. Muscle cells depolarized by the addition of 5.0 or 10.0 mM KCl to the bathing Ringer solution can be repolarized some 5 to 10 mV by the substitution of an equivalent amount of K-aspartate for KCl in the presence of 2.0 mM Mg⁺⁺. The repolarization produced by this method persists when the muscle is again placed in the initial KCl solution, thus eliminating the possibility that the hyperpolarization is due to the reduction of chloride in the bathing medium. If for some reason the resting membrane potential of the muscle fibers is considerably below (less negative than) the normal level of 92 mV reported for muscles bathed in 2.5 mM Ringer solution, the substitution of 2.5 mM K-aspartate for the 2.5 mM KCl and the addition of 2.0 mM Mg-aspartate to the Ringer solution will, within 15 minutes, repolarize the fiber to the normal level. Magnesium ions alone will not produce the observed repolarization nor can it be attributed to a reduction in the activity of the potassium in the Ringer solution.     

Sustained release of calcium elicited by membrane depolarization in ryanodine-injected mouse skeletal muscle fibers

The effect of micromolar intracellular levels of ryanodine was tested on the myoplasmic free calcium concentration ([Ca(2+)](i)) measured from a portion of isolated mouse skeletal muscle fibers voltage-clamped at -80 mV. When ryanodine-injected fibers were transiently depolarized to 0 mV, the early decay phase of [Ca(2+)](i) upon membrane repolarization was followed by a steady elevated [Ca(2+)](i) level. This effect could be qualitatively well simulated, assuming that ryanodine binds to release channels that open during depolarization and that ryanodine-bound channels do not close upon repolarization. The amplitude of the postpulse [Ca(2+)](i) elevation depended on the duration of the depolarization, being hardly detectable for pulses shorter than 100 ms, and very prominent for duration pulses of seconds. Within a series of consecutive pulses of the same duration, the effect of ryanodine produced a staircase increase in resting [Ca(2+)](i), the slope of which was approximately twice larger for depolarizations to 0 or +10 mV than to -30 or -20 mV. Overall results are consistent with the "open-locked" state because of ryanodine binding to calcium release channels that open during depolarization. Within the voltage-sensitive range of calcium release, increasing either the amplitude or the duration of the depolarization seems to enhance the fraction of release channels accessible to ryanodine.     

Voltage-dependent inactivation of slow calcium channels in intact twitch muscle fibers of the frog

Inactivation of slow Ca2+ channels was studied in intact twitch skeletal muscle fibers of the frog by using the three-microelectrode voltage-clamp technique. Hypertonic sucrose solutions were used to abolish contraction. The rate constant of decay of the slow Ca2+ current (ICa) remained practically unchanged when the recording solution containing 10 mM Ca2+ was replaced by a Ca2+-buffered solution (126 mM Ca-maleate). The rate constant of decay of ICa monotonically increased with depolarization although the corresponding time integral of ICa followed a bell-shaped function. The replacement of Ca2+ by Ba2+ did not result in a slowing of the rate of decay of the inward current nor did it reduce the degree of steady-state inactivation. The voltage dependence of the steady-state inactivation curve was steeper in the presence of Ba2+. In two-pulse experiments with large conditioning depolarizations ICa inactivation remained unchanged although Ca2+ influx during the prepulse greatly decreased. Dantrolene (12 microM) increased mechanical threshold at all pulse durations tested, the effect being more prominent for short pulses. Dantrolene did not significantly modify ICa decay and the voltage dependence of inactivation. These results indicate that in intact muscle fibers Ca2+ channels inactivate in a voltage-dependent manner through a mechanism that does not require Ca2+ entry into the cell.     

Calcium currents in a fast-twitch skeletal muscle of the rat

Slow ionic currents were measured in the rat omohyoid muscle with the three-microelectrode voltage-clamp technique. Sodium and delayed rectifier potassium currents were blocked pharmacologically. Under these conditions, depolarizing test pulses elicited an early outward current, followed by a transient slow inward current, followed in turn by a late outward current. The early outward current appeared to be a residual delayed rectifier current. The slow inward current was identified as a calcium current on the basis that (a) its magnitude depended on extracellular calcium concentration, (b) it was blocked by the addition of the divalent cations cadmium or nickel, and reduced in magnitude by the addition of manganese or cobalt, and (c) barium was able to replace calcium as an inward current carrier. The threshold potential for inward calcium current was around -20 mV in 10mM extracellular calcium and about -35 mV in 2 mM calcium. Currents were net inward over part of their time course for potentials up to at least +30 mV. At temperatures of 20-26 degrees C, the peak inward current (at approximately 0 mV) was 139 +/- 14 microA/cm2 (mean +/- SD), increasing to 226 +/- 28 microA/cm2 at temperatures of 27-37 degrees C. The late outward current exhibited considerable fiber-to-fiber variability. In some fibers it was primarily a time-independent, nonlinear leakage current. In other fibers it was primarily a time-independent, nonlinear leakage current. In other fibers it appeared to be the sum of both leak and a slowly activated outward current. The rate of activation of inward calcium current was strongly temperature dependent. For example, in a representative fiber, the time-to-peak inward current for a +10-mV test pulse decreased from approximately 250 ms at 20 degrees C to 100 ms at 30 degrees C. At 37 degrees C, the time-to-peak current was typically approximately 25 ms. The earliest phase of activation was difficult to quantify because the ionic current was partially obscured by nonlinear charge movement. Nonetheless, at physiological temperatures, the rate of calcium channel activation in rat skeletal muscle is about five times faster than activation of calcium channels in frog muscle. This pathway may be an important source of calcium entry in mammalian muscle.     

Swelling of the Müller fibers in the chicken retina

A high potassium concentration (33 meq) in the solution superfusing the isolated chicken retina causes an increase in the tissue transparency. An L-glutamate (1 mM) or L-proline (10 mM) solution has the same effect. Swelling of the Müller fibers, which have a radial position in the retina, could explain the transparency increase. This possibility was investigated in electron micrographs of retinas subjected to these treatments and fixed by freeze-substitution to preserve the water distribution in the tissue. The Müller fibers in the controls had a mean diameter of 0.22 micrometer. The fibers in retinas bathed for 2 min in the high [K+] solution were more than three times as thick (0.74 micrometer); the fibers in glutamate-treated retinas were more than twice as thick (0.49 micrometer). The fibers in the proline-treated retinas had a diameter of 0.39 micrometer. The glutamate- and proline-induced swelling may be due to a K+ release from neuronal elements, acting on the Müller fibers. The fiber swelling was postulated to be the expression of different Donnan equilibriums of fibers bathed in solutions of different K+ concentrations. The observed swelling caused by the high [K+] solution was compared with the theoretical swelling of the fiber as an ideal Donnan system, postulating permeabilities for different ions of the fiber membrane. This suggested that the high [K+] solution causes an increase in Na+ permeability in addition to the permeability of the membrane for K+, Cl-, and HCO3-. Chemical fixation with glutaraldehyde and formaldehyde in an Na-phosphate buffer yielded micrographs in which the Müller fibers of retinas treated with a high [K+] or a glutamate solution had diameters similar to those of the control preparations.