Na +-Ca 2+ exchange activity in synaptic plasma membranes derived from the electric organ of Torpedo ocellata

Abstract

Synaptic plasma membranes obtained by hypo-osmotic treatment of purified Torpedo ocellata synaptosomes, contain an electrogenic Na +-Ca 2+ exchange system. The dependence ofof the initial reaction rate on [Ca 2+] reveals a single binding site for Ca 2+ with an average apparent K m of 13.66 (S.D.= 12.07) μM [Ca 2+] and maximal reaction velocity of V max = 11.33 (S.D.= 5.93) nmol/mg protein per s. The dependence of the initial rate of the Na + gradient dependent Ca 2+ influx on the internal [Na +] exhibits a sigmoidal curve which reaches half-maximal reaction rate at 170.8 (S.D.=19.9) mM [Na +]. Addition of ATPγS does not change the K 0.5 to Na +. The average Hill coefficient is 3.09 (S.D.=0.86) indicating that 3–4 Na + ions are exchanged for each Ca 2+. Na + gradient dependent Ca 2+ uptake in Torpedo SPMs takes place also in the absence of K + suggesting that K + co-transport is not obligatory. The temperature dependence of the initial and steady-state rates of Na + gradient dependent Ca 2+ influx reveal that maximal reaction velocities of the Torpedo exchanger are attained between 15 and 20°C. The energy of activation between 0 and 20°C is 20 826 cal/mol. In comparison, rat brain synaptic plasma membrane Na + Na 2+ exchanger reaches maximal reaction rates between 30 and 40°C. Reconstitution of Torpedo or rat brain Na +-Ca 2+ exchangers into a membrane composed of either Torpedo or brain phospholipids, does not alter the temperature dependence of the native Torpedo or rat brain Na +-Ca 2+ exchangers; inspite of considerable differences in the composition of the fatty acyl chains that are esterified to brain and Torpedo phospholipid head groups and differences in membrane fluidity that were detected. An ATP-dependent Ca 2+ pump, which is insensitive to FCCP, is also present in the same synaptic membrane.