THE MECHANISM OF ION-TRANSPORT BY THE NA-CA2+,K+ EXCHANGE IN RODS ISOLATED FROM THE SALAMANDER RETINA()

1. Membrane currents caused by the operation of electrogenic Na+-Ca2+, K+ exchange were recorded from isolated rod outer segments under volt age-clamp using a whole-cell electrode. 2. Reversed mode exchange curr ents (Na(i)+-Ca(o)2+, K(o)+) were recorded with a high internal [Na+] and when both Ca2+ and K+ were present in the external solution. Omiss ion of either Ca2+ or K+ completely suppressed both the reversed excha nge current and the entry of Ca2+. 3. The charge transferred by the ex change per Ca2+ ion transported was identical in both forward and reve rsed modes. 4. The reversed exchange current declined as Ca2+ accumula ted inside the outer segment, and the form of this decline was consist ent with a first-order inhibition by internal Ca2+. 5. The reversed ex change current was increased e-fold by a 230 mV depolarization over th e range - 51 to + 29 mV. 6. The activation of reversed exchange by ext ernal Ca2+ was well described by first-order kinetics with a Michaelis constant, K(Cao)app, of 34 muM in the presence of 20 mm external K+. K(Cao)app was reduced by lowering external [K+], was increased by addi ng external Na+ and was unaffected by membrane potential.7. External K + also activated the exchange in a first-order manner with a Michaelis constant, K(Ko)app, of 151 muM in the presence of 0.5 mm external Ca2 +. K(Ko)app was reduced by lowering external [Ca2+], increased by addi ng external Na+ and was unaffected by membrane potential. 8. When the level of internal Ca2+ was increased via reversed exchange. K(Cao)app diminished in proportion to the reduction in the maximum current. but K(Ko)app remained approximately constant. 9. These observations cannot be reconciled with simple models of the exchange in which ions bind s imultaneously at opposite faces of the membrane before transport occur s. The results are broadly consistent with a consecutive model of the exchange in which unbinding of Na+ at either the external or the inter nal membrane surface is followed by binding of Ca2+ and then K+, and a re fully reproduced by a model in which Ca2+ binds before all the Nahas dissociated from the exchange molecule.