2 OPERATIONAL MODES OF K-CL COTRANSPORT IN LOW K-BLOOD-CELLS( SHEEP RED)

The present study further characterizes the two operational modes of K -Cl cotransport in low K+ sheep red blood cells previously described a nd proposed by us: (i) a membrane-bound cotransport entity devoid of m odulation by putative kinases/phosphatases, and (ii) an ATP-and Mg-mod ulated activity. We now report that quinine, spermine, and 300 mu M Ca -i(2+) inhibit (40-100%) K-Cl efflux in ATP-and Mg-i(2+) depleted cell s and hence interact directly with the cotransporter moiety. The sensi tivity of K-Cl cotransport to calyculin, however, was modulated by the ATP content of the cells: after depletion of both ATP and Mg-i(2+) th e flux was insensitive to the inhibitor, whereas at about 120 mu mol A TP/L cells calyculine inhibited K-Cl efflux by 95-100% in Mg-i(2+)-fre e cells. The K-Cl cotransporter itself, free of modulation by kinases/ phosphatases, 'senses' changes in cell volume perhaps at the membrane/ cytoskeleton level. Cellular ATP then switches the cotransporter's ope rational mode so that under physiological conditions the rate of K-Cl efflux is under the control of a putative kinases/phosphatases pathway .