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.
Animals Cell Line