EFFECTS OF OKADAIC ACID AND INTRACELLULAR CL- ON NA-K+-CL- COTRANSPORT()

The Na+-K+-Cl- cotransporter of the squid giant axon requires ATP and is inhibited by intracellular Cl- (Cl-i(-))in a concentration-dependen t manner ([Cl-](i) greater than or equal to 200 mM completely inhibits the cotransporter). In the present study we address the question of w hether inhibition of cotransport by Cl-i(-) is due to a Cl-i(-)-induce d increase of protein phosphatase activity. Intracellular dialysis was used to apply the phosphatase inhibitor okadaic acid (OKA) under cond itions of [Cl-](i) at 0, 150, or 300 mM during measurement of cotransp orter-mediated unidirectional Cl- influx into axone. At 0 mM [Cl-](i), the application of 250 nM OKA had no effect on the cotransport-mediat ed Cl- influx when axone were dialyzed with the normal intracellular A TP concentration ([ATP](i) = 4 mM). Reduction of [ATP] to 50 mu M resu lted in a significant decrease of the bumetanide-sensitive Cl- influx, which could be partially reversed by OKA treatment. Similarly, in ATP -limited axons with [Cl-](i) at 150 mM, cotransporter influx was parti ally stimulated by treatment with OKA. However, axons dialyzed with 30 0 mM [Cl-](i) ([ATP](i) = 50 mu M) had no measurable cotransport influ x, nor was subsequent treatment with OKA able to induce a cotransport- mediated Cl- influx. We conclude that the inhibition of cotransport ca used by Cl-i(-) is not the result of an increase in the OKA-sensitive protein phosphatase activity.