br Introduction When human red

Introduction When human red blood GANT 58 (hRBC) are suspended in depolarising Ringers, they respond by opening a non-selective voltage-dependent cation pathway, the NSVDC channel, which is permeable to mono- and divalent cations [1], [2], [3]. In patch clamp experiments on excised hRBC inside-out patches, bathed in salt solutions of physiological ion strength, depolarising potentials result in opening of a 30-pS channel [4]. At high positive potentials, +100 mV and above, the channel attains an open state probability of close to 1.0. However, the kinetics of the channel cannot be represented by a simple closed to open transition, since the open state probability depends not only on the instantaneous potential, but also on the prehistory of the channel [5], [6]. If the voltage is increased from deactivating (negative) potentials towards positive potentials, the open state probability is lower at a given potential, than compared to a change from positive potentials, at full activation, towards negative potentials. This hysteresis is thus a property of the individual channel unit. The number of channels in the intact hRBC has been estimated to be in the range of 150–300 [7]. Marked differences with regard to activation seem to exist compared to the isolated channel, but the hysteretic behaviour appears to persist as an ensemble property of the intact cells in suspension. Calcium added to the cells, following activation of the NSVDC-pathway by suspension in sucrose Ringers, results in a hyperpolarization due to activation of the Ca2+-activated K+ channels, the Gárdos channel, which deactivates the NSVDC channel. As a consequence of the deactivation, the Ca2+-influx ceases and the active extrusion of cellular calcium by the calcium-pump deactivates the Gárdos channel. Due to the closing of the conductive cation pathways, the conductive chloride pathway becomes more and more dominating, resulting again in positive membrane potentials, which in turn reactivates the NSVDC channel. In contrast to the original activation, from high positive potentials, the activation now takes off from negative potentials and the cation conductance becomes lower than before, even at more positive potentials [8]. In the following, a further characterization of the voltage activation and hysteretic properties at the level of the intact cell will be given.
Materials, methods and calculations
Results Initially, when cells are incubated in a sucrose Ringer in the absence of a chloride conductance blocker, the membrane potential has a value corresponding to the chloride Nernst potential (105 mV), which can be calculated from the intracellular Cl− concentration (100 mM) estimated from the membrane potential measured in n-Ringer. The NSVDC channels begin to activate and the membrane potential settles around 80 mV, from which it changes only slowly in the negative direction, due to the still dominating chloride conductance, see Fig. 1. When 10 μM of the chloride conductance blocker NS1652 is added, the rate of change of the membrane potential becomes faster and reaching either a peak or a stationary value after a period of time, depending on the lag time before addition of the chloride conductance blocker. The size of the hyperpolarization, reflecting the degree of cation conductance increase, depends too on the lag time, becoming bigger the longer the lag time, see Fig. 2. Adding calcium after the membrane potential has become constant results in a transient hyperpolarization, followed by a new stationary membrane potential, more positive than before the potential transient, see Fig. 3, signifying that the cation conductance is lower after the hyperpolarization. The hyperpolarization caused by addition of calcium is a graded response, depending on the calcium concentration and the initial depolarization, which was regulated by the degree of chloride substitution. Fig. 4 shows the difference in conductance after calcium hyperpolarizations compared to parallel experiments where Mg2+ was added instead of Ca2+, as function of the peak membrane potential of the hyperpolarization. It is seen that only negative peak potentials result in a conductance decrease after hyperpolarization. It should be noted, however, that the initial depolarization varies between the experiments, due to changes in the degree of substitution.