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

• Published in: The Journal of membrane biology Vol. 91; no. 3; p. 227
• Main Authors: Hoffmann, E K, Lambert, I H, Simonsen, L O
• Format: Journal Article
• Language: English
• Published: United States 01.10.1986
• Subjects: Animals; Biological Transport, Active - drug effects; Calcimycin - pharmacology; Calcium - pharmacology; Carcinoma, Ehrlich Tumor - metabolism; Cell Membrane Permeability; Chlorides - metabolism; Egtazic Acid - pharmacology; Female; Gramicidin - pharmacology; Ion Channels - drug effects; Ion Channels - metabolism; Kinetics; Mice; Mice, Inbred Strains; Pimozide - pharmacology; Potassium - metabolism; Quinine - pharmacology; Thermodynamics; Valinomycin - pharmacology
• ISSN: 0022-2631
• DOI: 10.1007/bf01868816

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 of 42K and 36Cl tracer fluxes is directly demonstrated. RVD and the A23187-induced cell shrinkage both are: inhibited by quinine which blocks the Ca2+-activated K+ channel, unaffected by substitution of NO-3 or SCN- for Cl-, and 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 by 42K 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 from 42K 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.