Separate, Ca2+-activated K+ and Cl− transport pathways in Ehrlich ascites tumor cells

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Abstract
The net loss of KCl observed in Ehrlich ascites cells during regulatory volume decrease (RVD) following hypotonic exposure involves activation of separate conductive K+ and Cl− transport pathways. RVD is accelerated when a parallel K+ transport pathway is provided by addition of gramicidin, indicating that the K+ conductance is rate limiting. Addition of ionophore A23187 plus Ca2+ also activates separate K+ and Cl− transport pathways, resulting in a hyperpolarization of the cell membrane. A calculation shows that the K+ and Cl− conductance is increased 14-and 10-fold, respectively. Gramicidin fails to accelerate the A23187-induced cell shrinkage, indicating that the Cl− conductance is rate limiting. An A23187-induced activation of42K and36Cl tracer fluxes is directly demonstrated. RVD and the A23187-induced cell shrinkage both are: (i) inhibited by quinine which blocks the Ca2+-activated K+ channel. (ii) unaffected by substitution of NO 3 − or SCN− for Cl−, and (iii) inhibited by the anti-calmodulin drug pimozide. When the K+ channel is blocked by quinine but bypassed by addition of gramicidin, the rate of cell shrinkage can be used to monitor the Cl− conductance. The Cl− conductance is increased about 60-fold during RVD. The volume-induced activation of the Cl− transport pathway is transient, with inactivation within about 10 min. The activation induced by ionophore A23187 in Ca2+-free media (probably by release of Ca2+ from internal stores) is also transient, whereas the activation is persistent in Ca2+-containing media. In the latter case, addition of excess EGTA is followed by inactivation of the Cl− transport pathway. These findings suggest that a transient increase in free cytosolic Ca2+ may account for the transient activation of the Cl− transport pathway. The activated anion transport pathway is unselective, carrying both Cl−, Br−, NO 3 − , and SCN−. The anti-calmodulin drug pimozide blocks the volume- or A23187-induced Cl− transport pathway and also blocks the activation of the K+ transport pathway. This is demonstrated directly by42K flux experiments and indirectly in media where the dominating anion (SCN−) has a high ground permeability. A comparison of the A23187-induced K+ conductance estimated from42K flux measurements at high external K+, and from net K− flux measurements suggests single-file behavior of the Ca2+-activated K+ channel. The number of Ca2+-activated K+ channels is estimated at about 100 per cell.