Kinetics and energetics of calcium efflux from intact squid giant axons.

Abstract

The Ca efflux from intact squid [Loligo vulgaris, L. forbesi] axons consists of 3 major components: 1 activated by Cao [external Ca] 1 activated by Nao [external Na] and a residual flux that persists in the nominal absence of both Cao and Nao. The properties of these components are investigated in unpoisoned axons and in axons poisoned with cyanide. Under all conditions the shape of the curve relating Cao to Cao-activated Ca efflux approximates to a section of a rectangular hyperbola, consistent with simple Michaelis activation. The external Ca concentration giving half-maximal activation of Cao-activated Ca efflux is .apprx. 2.mu.M in unpoisoned axons immersed in Na-ASW, but on poisoning changes progressively to values in the range 1-10 mM. The residual efflux from unpoisoned axons may reflect activation by traces of Ca present immediately external to the axolemma. The apparent affinity for Cao of Cao-activated Ca efflux is very similar in unpoisoned axons immersed in sea waters containing Na, Li, Tris or K as major cation, whereas in poisoned axons the affinity in Na and Li is about the same but higher than that in choline and Tris. In unpoisoned axons Ca influx increases linearly as Cao is increased from 2 .mu.M-110 mM. The absolute value of the Ca influx from 10 .mu.M-Cao is < 1% of the Cao-activated Ca efflux at this external Ca concentration. In poisoned axons the sizes of Cao-activated Ca efflux and Ca influx were similar at all Ca concentrations examined. The shape of the curve relating Nao to Nao-activated Ca efflux approximates to a section of rectangular hyperbola in unpoisoned axons but is clearly sigmoidal in axons fully poisoned with cyanide. The sigmoidal shape develops progressively during poinsoning. The external Na concentration giving half-maximal activation of Nao-activated Ca efflux is .apprx. 60 mM in unpoisoned axons and often > 300 mM in fully poisoned axons. In unpoisoned and poisoned axons at constant Nao, replacement of 200 mM-Tris by 200 mM-K reduces the Nao-activated Ca efflux reversibly. The magnitude and kinetics of both Cao-activated Ca efflux and Nao-activated Ca efflux are unaffected by injection into the axon of a solution containing 220 mM-EGTA and 100 mM-CaCl2. Subsequent exposure to cyanide reduces the amplitude of the Ca efflux to .apprx. 1/3, and the kinetics of both components of the Ca efflux changes to that characteristic of the poisoned state. Injection of ATP produces a transcient increase in efflux and restores a Nao-activation curve close to that seen in unpoisoned axons, AMP and cyclic AMP are ineffective. Application of cyanide in the presence of oligomycin produces a large increase in Ca efflux. Subsequent injection of ATP has little effect on the size of the efflux but alters the kinetics of Nao-activation so that they resemble more closely those seen in unpoisoned axons. Results are consistent with a model in which the Ca pump must bind ATP for efflux to occur.