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  • In order to test the influence of the initial


    In order to test the influence of the initial depolarization, experiments were performed, where the degree of chloride substitution was varied, either by adding n-Ringer to KN-62 incubated in a sucrose Ringer or by suspending the cells directly in Ringers identical to the final composition in the first series of experiments. The resulting cation conductances vs. the stationary membrane potentials are shown in Fig. 5. Fig. 6 shows the conductances vs. the final chloride Nernst potentials. As demonstrated in Fig. 6, the cation conductances calculated for cells preincubated in sucrose Ringer was consistently higher than for cells, which had been directly injected into solutions with higher Cl− concentrations, and consequently initially less depolarized.
    Discussion A major obstacle for the comparison of results from patch clamp studies with experiments on voltage-activated channels in intact cells in suspension is the lack of control with regard especially to the membrane (clamp) potentials. As demonstrated both for the isolated channel [9] and the intact cells in suspension, the conductance changes caused by activation of the NSVDC channel are a function of both time and potential (Fig. 2, Fig. 5, open symbols). Initially, when human red cells are suspended in sucrose Ringers containing only low concentrations of KCl, the membrane potential attains a value identical to the positive chloride Nernst potential. Over time, the membrane potential changes towards more negative values, due partly to activation of the NSVDC channels and partly to the dissipation of the potassium and chloride gradients. With a normal chloride conductance of about 20 μS/cm2, the salt loss from the cells is considerable, although the membrane potential is fairly constant (quasi-voltage clamp). With the chloride conductance reduced to about 2 μS/cm2 in the presence of 10 μM NS1652 [11], the salt loss is reduced (quasi current clamp), but the membrane potential changes in the negative direction relatively fast. The experiments where the cells are incubated in sucrose Ringer, and the chloride conductance blocker is added after a lag time (Fig. 1), show that the longer the cells have been ‘voltage-clamped’ the higher the resulting cation conductance. It should be noted, however, that the increased activation is ‘remembered’, since it results in a more negative membrane potential, which should be deactivating. Compared to patch clamp experiments [6], [9], which were, however, done at room temperature and at higher salt concentrations, the time course seems to be somewhat slower, but of the same order of magnitude. With regard to the deactivation, the difference is pronounced. The isolated channel deactivates after an instantaneous jump to negative potentials, with a halftime of about 15 ms [9], whereas the activated channels in the intact cell remain open for minutes, even at moderately negative potentials, and only at very negative potentials they seem to begin to deactivate. Very negative transient membrane potentials can be attained by addition of Ca2+ to cells where the NSVDC channels have been sufficiently activated to allow Ca2+ to permeate, thereby selectively increasing the potassium conductance further by activation of the Gárdos channel (see Fig. 3). The hyperpolarization to −75 mV indicates that no appreciable degradation of the chloride and potassium gradients have occurred during the voltage clamp period. However, since the Nernst potential changes due to the degradation are in opposite directions, the calculation is relatively insensitive to a moderate loss of cellular KCl. This hyperpolarization seems to deactivate the NSVDC channel, thus decreasing the Ca2+-entry concomitant with a delayed [12] active extrusion of cellular calcium by the Ca2+-pump, leading to deactivation of the Gárdos channel. Since the total cation conductance decreases during this sequence of events, the cells are depolarized again due to the persistent positive chloride Nernst potential. This in turn reactivates the NSVDC channel, but in a different state of activity, with a lower conductance reflected in a more positive potential than before the potential transient (Fig. 3). This mode of operation seems to be a parallel to the hysteretic behaviour of the isolated channel, where a plot of the open state probability vs. the clamp potential has two ‘legs’: a low activity state when the clamp potential goes from negative towards positive values and a high activity state going from positive to negative values. This is further supported by differences in conductance seen at identical final sucrose substitutions, but resulting either from activation from very positive membrane potentials showing a high activity compared to the lower activity seen when the channel has been activated at less positive potentials. It is apparent from Fig. 5 that there is no simple correlation between the NSVDC channel conductance and the stationary membrane potential. If, however, the channel conductance is plotted against the final chloride Nernst potential (Fig. 6), the two activity states become evident. Although not a hysteresis curve proper, since the activity is plotted against the resulting chloride Nernst potential, Fig. 6 reflects the memory effect at the cellular level.