br Acknowledgments br Introduction When human red blood cell
Introduction When human red blood cell (hRBC) membranes are depolarized, either as intact cyclic amp function suspended in depolarizing (low Cl−) Ringers or under voltage clamp conditions using excised inside-out patches, they respond by opening a nonselective voltage-dependent cation (NSVDC) channel which is permeable to mono- and divalent cations , , , , , . The NSVDC channel constitutes, together with the erythrocyte Ca2+-activated K+ channel, the Gárdos channel, also known as hIK ,  and hSK4 , and the Ca2+-ATPase, a feedback-regulated system which at least under experimental conditions becomes operative . The addition of Ca2+ to a suspension of red cells with fully activated NSVDC channels, which is achieved after 5–8 min of incubation in a sucrose-substituted Ringer containing 2 mM KCl, causes a transient hyperpolarization due to activation of the Gárdos channel. The transience of the Gárdos channel activation is ascribed to a combined effect of the voltage-dependent deactivation of the NSVDC channel in response to the hyperpolarization, causing the Ca2+ influx to terminate, and a delayed activation of the Ca2+ pump extruding the calcium again . The Gárdos channel activation caused by the Ca2+ influx through the NSVDC channel can pharmacologically be suppressed by the addition of the established blockers cetiedil , nitrendipine , , or clotrimazole . Although the Gárdos channel activation is abolished by all these compounds, unexpectedly it was found that clotrimazole, at concentrations above that needed for full Gárdos channel block, potentiated the voltage- and time-dependent activation of the NSVDC channel. In the present paper, we pursue the NSVDC effect of clotrimazole and other Gárdos channel blockers.
Materials, methods, and calculations
Results When red cells are injected into sucrose-substituted Ringer solution containing the Cl− conductance blocker NS1652, the initial very positive potential is followed by a slow repolarization towards 0 mV during the next 300–500 s (Fig. 1) reflecting the time-dependent activation of the NSVDC channel. When activation is fully developed, indicated by a stationary membrane potential, addition of 1 mM Ca2+ initiates a fast and pronounced hyperpolarization reaching about −80 mV within 60 s. As previously reported , this hyperpolarization is due to activation of the Ca2+-activated K+ channel via Ca2+ influx through the NSVDC channel. In the presence of 20 μM clotrimazole, the initial repolarization reached a more negative plateau (−35 mV) and occurred faster than the control experiment, indicating a higher degree of activation of the NSVDC channel. As expected, the addition of Ca2+ in this situation did not result in a Gárdos channel-mediated hyperpolarization due to the blockage of this channel. In nR, clotrimazole inhibits the Ca2+/A23187-induced hyperpolarization with a half inhibition concentration IC50 of 79 nM (not shown). In accordance with the notion that clotrimazole activated the cation conductance, it was found that the potassium net efflux increased too (see Table 1). The K+ conductances were calculated from Eq. (2), and an increase of about 70% was found at 25 μM clotrimazole. When clotrimazole was added after the NSVDC channel had attained a stationary activation (Fig. 2), a fast hyperpolarizing shift in membrane potential occurred showing that the activation is fast and that the activated channel is sensitive to modulation. In contrast to clotrimazole, nitrendipine (IC50 = 270 nM) and cetiedil (IC50 = 50 μM), which are both Gárdos channel inhibitors of different structural classes, had no effect on the NSVDC channel activation. For comparison, a typical example with nitrendipine is shown in Fig. 2 and dose-response data are shown for cetiedil in Fig. 3. These results demonstrate that the activation of the NSVDC channel is not linked to the block of the Gárdos channel per se.