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

Summary

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.