Volume sensitive 36Cl fluxes in L6 myoblasts.

Efflux of 36Cl ions was followed in undifferentiated L6 myoblasts at rest and after exposure to hypotonic saline in order to test participation of the chloride exit in regulatory volume decrease mechanism. The efflux curve can be fitted at least with two time constants. After the hypotonic stress the slow rate constant increases almost threefold, from 0.021 +/- 0.019 min-1 normal saline to 0.060 +/- 0.019 min-1 in hypotonic solution.

A note on the cellular effects of nystatin in single myoballs.

Volume changes in single L6 myoblasts (myoballs) exposed to nystatin solutions were followed on single cell level by means of quantitative video image analysis. The myoblasts swelled in nystatin solutions. The volume change was dependent on the nystatin concentration, the threshold concentration being 12.5 mumol/l of nystatin freshly dissolved in Krebs solution. The threshold effect was triphasic: a slight initial volume decrease (shrinkage) for about 2 min followed by a volume increase and, after about 10 min by a significant volume decrease. At twice as high nystatin concentration (25 mumol/l) the final shrinkage phase was lacking. At 50 mumol/l concentration the volume increased continually after a delay of about 1-2 min. and reached a plateau of about 350% of the original volume. At 100 mumol/l concentration of nystatin the myoblasts increased their volume in about five min to more than 500% of the original value. The effects of nystatin diminished upon prolonged storage of nystatin Krebs solution. Nystatin solutions (50 mumol/l) prepared 3 hours before use were stil active to about 80%. Volume changes in 100 mumol/l nystatin solutions were, however, substantially diminished (to about 20%) 5 hours after the preparation of the nystatin solution. By replacing external Na+ by TEA+ in the presence of external Cl- a regulatory volume decrease was observed to subnormal values; the myoblast volume shrank to about half of the control value. The volume changes were reversible after reintroduction of Krebs solution. The regulatory volume decrease to subnormal values was also observed after replacing external Cl- by glutamate anion in the presence of external Na. The volume changes were, however, not reversible after reintroduction of Krebs solution. The swelling of myoblasts in 50 mumol/l nystatin Krebs solution continued after a definite enlargement of the whole myoblast was reached with the formation of several blebs, which eventually coalesced to form a continuous layer around the myoballs. The enlarged vesicles in nystatin solutions were able to start and fulfill the mitotic cycle. Cell volume measurements represent a handy means for checking the activity of nystatin solutions for the perforated patch experiments.

Regulatory volume decrease in cultured myoblasts L6.

A method is described for measuring volume changes in single L6 myoblasts at a stage of proliferating "myoballs", which allows to follow volume changes on single cell level by means of quantitative video image analysis. Myoblasts exposed to hypoosmotic or hyperosmotic challenges for up to 3-5 min behave as osmometers. The relative cell volume is a linear function of the reciprocal of the relative osmolality in the range 0.5-2T. Cells exposed to hypotonic Krebs solution with Na+ and Cl- ions as the main ions exhibit volume readjustment towards the original level. The regulatory volume decrease (RVD) was complete after about 15 min in hypotonic solution with Cmax (maximum RVD) increasing with the decrease in osmolality in the test solution. By replacing external Na+ by K+ in the presence of external Cl- regulatory volume decrease was reversed; myoblast volume continued to increase. RVD was present after replacing Cl- with NO3. Quinine (0.5 mmol/l) partially blocked RVD. It is suggested that RVD in L6 myoblasts is mediated mainly by separate K+ and Cl- channels.

Selectivity of maxi chloride channels in the L6 rat muscle cell line.

A method is described for the determination of reversal potentials (EREV) from variance of single-channel currents vs. membrane potential in ramp pulse mode. The variance-voltage relationship is represented by a parabola with a minimum of the best fit curve corresponding to the value of EREV. The reversal potential of the maxi-Cl channels changes according to the activity of Cl ions at the internal side of the excised (inside-out) patch membrane as expressed by the Goldman-Hodgkin-Katz equation with PA/PCl = 0.15, indicating the anion nature of the channel. The relative permeabilities (PA/PCl found for halide anions, were 1.15 for iodide and 1.18 for bromide. The relative permeabilities measured (PA/PCl) for other anions were 1.13 for nitrate, 0.59 for bicarbonate, 0.60 for methanesulfonate, 0.40 for SO42-, 0.44 for propionate, and 0.10 for glutamate. No significant differences in PA/PCl of the investigated anions were observed between proliferating myoballs and quiescent myoblasts. This may mean that the newly formed channels possess full-grown selective filters. A close correlation (r = 0.89) was found between the calculated Stokes diameters of the anions under investigation and their relative permeabilities (PA/PCl). The intercept of the best fit line with the abscissa is 7.2 A (7.2 x 10(-10)m), which may correspond to the diameter of the selectivity filter of the maxi-chloride channel in L6 myoblasts. A similar value of the channel size was obtained from the relationship between the minimum cross-sectional areas of the anions and their relative permeabilities, PA/PCl. The best fit line intercepts the abscissa at 27.5 A2, indicating a pore size approximately 6 A. The minimum areas were obtained by computer from molecular models of the anions.

Conductance-voltage relations in large-conductance chloride channels in proliferating L6 myoblasts.

Large-conductance chloride channels (maxi-Cl channels) were studied in cultured myoblasts (L6 rat muscle cell line); in excised (inside-out) and in cell attached membrane patches using a conventional patch clamp method. The incidence of maxi-Cl channels was substantially higher in proliferating myoballs, then in quiescent (bottom-attached) myoblasts (90% and 50% percent of examined cells, respectively). The maxi-Cl channels in myoballs were present both in cell attached and excised patches. The channel conductance at symmetric [Cl] = 150 mmol/l was 359 +/- 42 pS (n = 74) in quiescent cells and 439 +/- 10 pS (n = 6) in proliferating myoballs respectively. The conductance of the channel in quiescent cells increased with chloride concentration in symmetric NaCl rich solutions according to Michaelis-Menten curve with the saturation limiting conductance of about 640 pS (gmax) and Km = 112 mmol/l. The shift of the reversal potential upon increasing the pipette concentration of NaCl from 150 to 250 mmol/l was consistent with PNa/PCl = 0.1. Neither the conductance nor the activation of the channel were dependent on the presence of calcium ions. The bell-shaped steady state channel conductance-voltage relationship is asymmetric and can be fitted by two Boltzmann equations with different Vh and k constants; -25.6 mV and -6.8 mV, respectively, for the negative side and +49.6 mV and +13.7 mV for the positive side in quiescent cells. The corresponding values in proliferating myoballs were as follows: -15.5 mV and -2.4 mV, respectively, for the negative side and +31.4 mV and +6.8 mV for the positive side. From the maximum slopes of the Popen versus V curves an estimate was made of the charges for the gates that close at negative (3.5) or positive (1.7) potentials, respectively, in quiescent cells. The corresponding values in myoballs were 10.6 and 3.7, respectively. The probability of one gate to be open was dependent on the state of activation of the opposite gate as determined by prepulses of the opposite polarity. The channel showed multiple (up to six) conductance levels that may develop in a step-like manner. The onset of the full-grown maxi-Cl channel is fairly abrupt; it might, however, be preceded by a small conductance unit activity. It is supposed that the differences between the quiescent myoblasts and proliferating myoballs might reflect increased expression of maxi-Cl channels in myoballs to perform as yet unknown role in the cell cycle and/or proliferation of the myoblasts.

Arachidonic acid blocks large-conductance chloride channels in L6 myoblasts.

Modulation of high-conductance chloride channels by eicosanoids, stilbene derivatives and Zn2+ ions was studied in cultured myoblasts (L6 rat muscle cell line) in excised and cell-attached membrane patches using a conventional patch clamp method. Arachidonic acid (AA) blocked the channel at concentrations 1-50 mumol/l from the internal side of the membrane in excised (inside out) patches with a time constant of about 20 s. The block was absent when arachidonic acid was applied to the bathing solution in cell attached patches, or to the pipette solution, respectively. Arachidonic acid changed the probability of the channel being in the open state (Popen) in dependence on the applied voltage (V). The asymmetric bell-shaped Popen - V relationship showed a steeper dependence on both the negative and positive voltage, respectively, in arachidonic acid solutions. The midpoint potentials (Vh) of the Popen - V relationship were shifted towards lower membrane potential displacements from the holding potential. The recovery from the block was very slow but was found to be enhanced by application of a symmetric voltage ramp pulse. The stilbene derivative (DIDS) blocked the channel at mu molar concentrations (10-100 mumol/l) applied from the internal side of the membrane. The onset and recovery of the DIDS block showed characteristics similar to the AA block. The channel was found to be blocked reversibly by short application of Zn+ ions (1-10 mmol/l) from the internal side of the membrane.

High-conductance chloride channels in BC3H1 myoblasts.

The existence of a high-conductance voltage sensitive chloride channel in BC3H1 myoblasts is documented. The conductance of the channel in symmetrical 150 mmol/l sodium chloride is around 400 pS. The probability of the channel being in the open state decreases with increasing of the imposed voltage from holding potential (0 mV) in both the depolarizing and the hyperpolarizing direction respectively. The bell-shaped open probability plot is asymmetrical, and can be fitted by two Boltzmann equations with different Vh and kn constants; the fitted values were -53 mV and -8.4 respectively for the negative side and +41 mV and +10.2 for the right side. When the unit Cl currents to rectangular pulses are summated the resulting total ionic Cl current shows relaxation, which increases with the amplitude of the pulse. The activation as well as the shape of the current can be significantly influenced by varying the amplitude and the direction of prepulses or holding potential. The high-conductance Cl channel shows several substrates (at least four with amplitudes of around 100 pS); frequently accompanied by flickerings.

Semi-automatic analysis of unit channel conductance from voltage ramp records.

An IBM PC-compatible computer program, RAMP, for evaluation of single-channel recordings acquired using voltage ramp protocols is presented. The program uses semi-automatic procedures to make necessary corrections to a record (e.g. subtraction of baseline shift) and to measure all channel slope conductances as well as reversal potentials. The output is either a hardcopy of graphic display, which includes the calculated parameters, or data in ASCII format for further use (e.g. plots using various graphic software). Originally, the software was developed for the evaluation of voltage ramp records of single channel data from maxi chloride channels in myoblasts of a muscle cell line (Hurnák and Zachar 1992). Records from these membrane patches were also used in this work to demonstrate basic principles of the software and its practical use in evaluating single channel records obtained in response to the application of voltage ramps. The channel conductances calculated from ramp records were compared with those obtained by classical evaluation procedures from voltage step records.

Maxi chloride channels in L6 myoblasts.

The existence of a large conductance voltage sensitive chloride channel is documented in undifferentiated cells (myoblasts) of the L6 rat muscle cell line. At this stage of development the resting membrane conductance is dominated by potassium ions only (Kidokoro 1975). The conductance of the channel in symmetrical 120 mmol/l choline chloride is 331 +/- 4 pS. The probability of the channel being in the open state decreases with the increasing imposed voltage. Due to rapid inactivation at high membrane potential deviations (both negative and positive) from the equilibrium potential the channel can be resolved clearly by pulse technique protocols only. The incidence of the channel in successful patch trials was higher than usually reported. The channel was present after differentiation of the myoblasts into the myotubes. It showed at least one definite substate and pronounced flickerings between the substate and the main open state. The channel was observed in myoblast attached patches as well. It is supposed to belong to the category of maxi chloride channels, and to play probably a role in regulatory volume readjustment or in cell communication during myogenesis, respectively.

Low-conductance chloride channel from crayfish skeletal muscle incorporated into planar lipid bilayers.

Low-conductance chloride channel from skeletal muscle SR vesicles of the crayfish Astacus fluviatilis was incorporated into planar lipid bilayers and its basic characteristics were investigated. The channel has a relatively low unitary conductance of 26 pS in symmetrical 160 mmol/l choline-chloride. The dependence of the channel conductance on Cl- concentration shows saturating behavior with a maximum conductance of 37 pS and an ionic activity for half-maximum conductance Km = 75 mmol/l. The channel exhibits a complex kinetics with several modes of activity. Open state probability slightly decreases with the increasing absolute value of voltage. The channel activity does not appear to be dependent on the presence of Ca2+ ions. The channel is effectively inhibited by DIDS, a stilbene derivative. The permeability properties of the channel are similar to the specific behavior of the "double-barrelled" channel from Torpedo electroplax described by Miller and White (1984).

Ryanodine receptor purified from crayfish skeletal muscle.

The ryanodine receptor was isolated from the sarcoplasmic reticulum of crayfish skeletal muscle. Ryanodine binding to the native fraction was measured by Scatchard analysis and values of 60 nmol/l and 9 pmol/mg were obtained for KD and Bmax respectively. The identity of purified receptor was confirmed by electron microscopy, electrophoresis and incorporation into planar lipid bilayers. At least two conductance states (100 pS and 50 pS) were observed in 100 mmol/l NaCl both for native and purified receptor.

The Ca2+ antagonists binding cytosolic protein has properties of the Ca2+ channel.

In our previous work (Krizanová et al. 1989) we have described a protein from rabbit skeletal muscle cytosolic fraction, which is able to bind dihydropyridines and phenothiazines. In the present work conclusive evidence is provided for the ability of the phospholipid-reconstituted cytosolic protein to transport calcium. The calcium transport was stimulated by BAY K 8644 and inhibited in the presence of PN 200-110. Our observations were confirmed also by electrophysiological measurements on planar lipid bilayers. The possibility that the cytosolic fraction was contaminated with membranes could be definitely ruled out. Nevertheless, the nature of the protein under study is still in the frame of guess.