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  • The observed increase in macroscopic conductance


    The observed increase in macroscopic conductance could be due to a left shift in the open state probability vs. voltage curve for the high-activity state causing an increase in the open state probability at all potentials. However, an increase of the apparent single channel conductance cannot be entirely excluded, for example, due to a changed distribution between the numerous conductance states described for this channel [18]. This question, however, cannot be answered by experiments on cell suspensions, but only by single channel experiments. The molecular identity of the NSVDC channel is presently unknown, although the recent description of a genuine voltage-dependent member of the transient receptor potential 2076 [19] is inspiring and may suggest a relation to this diverse and fast-growing family of cloned cation channels. The emergent pharmacological profile described here may help identifying the gene encoding the human erythrocyte NSVDC channel.
    Introduction Under physiological or near physiological conditions, the concentration of free Ca2+ in the red cell cytosol is maintained at a very low level, probably about 30 to 60 nM [1], [2] by the powerful CaATPase [3], thereby maintaining the activity of the Ca2+ activated K+ channel, the Gardos channel, very close to zero. It has been shown that the passive calcium influx, which if not countermanded would activate the Gardos channel, can be described by a saturating and a linear component, where the linear component is negligible in fresh cells. The saturating component has been described by a ‘hyperbole’ with K0.5 about 1 mM, and an influx at this extracellular concentration of 50 μmol/(lcell h) [4], [5]. Trans-acceleration was observed, and it was concluded, that a major part of the Ca2+ influx, under physiological conditions was carrier mediated. A serious problem for the characterization of the passive Ca2+ influx, especially at low extracellular Ca2+ concentrations, is the lack of specific CaATPase (PMCA) inhibitors. Commonly used methods have been metabolic depletion or vanadate treatment. However, in both cases a residual pumping activity remains [6] and furthermore vanadate seems to increase the passive Ca2+ influx [7], [8]. In the intact red cell, the Ca2+ sensitivity for activation of the PMCA and the Gardos channel is of the same order of magnitude, in the range 0.5 to 1.0 μM. However, in recent years, a number of Gardos channel agonists have become available [9], [10], and one of these, NS309 seems up to now to be the most powerful with regard to a decrease of the K1/2(Ca2+) for the Gardos channel. In the present work, NS309 has been used to hypersensitize the Gardos channel, causing activation at a, possibly subphysiological, Ca2+ level, where the PMCA can be assumed to be almost inactive [5]. In a low potassium Ringer, the Gardos channel activation resulting from a spontaneous Ca2+ influx will then cause a hyperpolarization due to the K+ conductance increase, and the concomitant loss of KCl and water will cause the cells to shrink, increasing the density and the osmotic fragility.
    Materials and methods
    Results Following injection of the packed red cells into the unbuffered experimental medium (nR), the extracellular pH settles at a value corresponding to the normal red cell membrane potential of about −10 mV. Immediately following addition of NS309, the membrane potential hyperpolarizes dose-dependently (not shown). At 100 μM NS309, the membrane potential hyperpolarizes to about −80 mV, corresponding to a K+ conductance of about 5 μS/cm2, a hyperpolarization which can be reversed by addition of 10 μM nitrendipine, see Fig. 1, and clotrimazole (not shown). It should 2076 be noted, that the potential response is enhanced, since the chloride conductance is blocked about 90% in the presence of 10 μM NS1652. In the presence of 10 μM NS309, the induced hyperpolarization showed a marked dependence on the extracellular calcium concentration, with the hyperpolarization reaching the same level at 1 mM Ca2+ as seen with 100 μM NS309 in the presence of only contaminating amounts of Ca2+ (about 4 μM). However, the hyperpolarization following NS309 addition, is relatively slow at all concentrations of [Ca2+]ex compared to the hyperpolarization induced by the action of the calcium ionophore A23187 at the lowest [Ca2+]ex, see Fig. 2.