The waiting time for first arrival of cells
The waiting time for first arrival of 77 into the dense fraction of 3–4 min at the normal chloride conductance, and about 30 min with inhibition to about 10% of the normal value, signifies that the Gardos channels in the activated cells become maximally or near maximally activated, corresponding to about 60 to 80 μS/cm2 , . Since not all cells are activated instantaneously, as is the case under the action of NS309, the intracellular free calcium concentration in the major fraction of the cells must be too low, due to the active extrusion, to cause Gardos channel activation, even in the presence of NS309. It has been shown, that the Gardos channel activation induced by A23187 is instantaneous and near maximal. As can be seen from Fig. 2, the activation caused by NS309 results in an apparent hyperpolarization, which is lower and develops more slowly. The probabilistic activation by NS309 of the individual cells, as demonstrated in Fig. 4 explains the phenomenon. If the cells, instead of being homogeneously activated, are either activated, which means that they hyperpolarize, or remains in the resting state, the change in the extracellular pH cannot be used directly for a membrane potential calculation, but only in a qualitative sense. The cells with maximally activated Gardos channels will hyperpolarize to about −90 mV respectively −108 mV at gCl ∼2 μS/cm2. The cells which remain in the resting state are equivalent to a buffered extracellular medium relative to the activated cells, and changes in the cellular pH will occur, both for the cells in the activated and resting state . The membrane potential estimated from the suspension pH thus becomes a weighted mean value, where the weighting factors are the fractions in the respective states. Although the average Ca2+ influx has been shown to be dominated by a carrier like mechanism, infrequent random opening of a calcium entry pathway in the individual cell, could lead to an abrupt increase of the [Ca2+]cell, causing the Gardos channels in this cell to open, whereby the membrane potential hyperpolarizes. However, the lag times for the first arrival of cells in the dense state indicate that a cell, once activated stays activated for 4 or 30 min, which at maximum Gardos channel activation is necessary for the cell to reach a density above 1.118 g/ml cells. This seems to imply, that the resting pump-leak cellular Ca2+ concentration is below the threshold for Gardos channel activation, but following a Ca2+ burst the PMCA cannot lower the Ca2+ concentration sufficiently to deactivate the Gardos channel again, as directly seen with cells suspended in sucrose Ringer (Fig. 3). Tentatively this might be ascribed to the establishment of a new steady state at a higher concentration, due to the hyperpolarization. It should be noted, that since the Gardos channel activation probably is dependent on at least the 2nd power of the [Ca2+]cell, as has been shown for IK and SK channels ,  a lowering of the half activation concentration for Gardos channel activation to about 50 nM gives a very steep activation curve, almost resembling a square function. At present, it is unclear, why the fraction of cells recruited to the high resistance state at infinity seems to be dependent on the extracellular calcium concentration. A number of explanations can be guessed at: 1) a distribution over the cell population of the Gardos channel calcium sensitivity, 2) a distribution over calcium permeability and 3) a distribution over PMCA activity at low or subphysiological calcium concentrations. The most probable explanation seems to be the distribution over the pump activity. It has been shown, that the Vmax for the pump covers a wide range of values, from about 5–35 mmol (340 g Hb)h . If this spread in Vmax is paralleled by the pump rates at physiological or subphysiological [Ca2+]in the increased influx at an elevated [Ca2+]out once a ‘calcium channel’ opened would lead recruitment of the cells with the highest pump rates, too.