Differential up-regulation of Na+-Ca2+ exchange by phosphoarginine and ATP in dialysed squid axons

The aim of this study was to characterize further the two main metabolic pathways of regulation of the Na + -Ca 2+ exchanger in squid axons induced by its two naturally ocurring high-energy compounds: ATP and phosphoarginine (Pa). [Na + ] o -dependent Ca 2+ efflux (forward - exchange) and [Ca 2+ ] o -dependent Ca 2+ efflux ( - exchange) were measured in internally dialysed squid axons at 16–17 °C. Measurements of changes in the apparent affinity of the Na + -Ca 2+ exchanger for transporting ( , , , ) and regulatory ( ) ions induced by ATP and Pa show marked differences for the two substrates: (i) ATP strongly alters the affinity for and , while Pa does not, and (ii) in the absence of , ATP has no stimulatiory effect; on the other hand, Pa causes a dramatic increase in - exchange with little activation of - exchange. The MgATP analogue chromium-ATP (CrATP) completely inhibits MgATP stimulation of the Na + -Ca 2+ exchanger. Nevertheless, even with the effects of the nucleotide blocked, Pa exhibits its usual activation of the [Na + ] o -dependent Ca 2+ efflux. None of the classical serine-threonine-tyrosine kinase inhibitors, nor the PP 1 and PP 2 phosphatase inhibitors, affects either the ATP or the Pa effect. However, intracellular microinjections of an exogenous phosphatase (alkaline phosphatase) completely reverses the stimulation of the Na + -Ca 2+ exchange induced by ATP and Pa. Prolonged intracellular dialysis with highly permeable porous capillaries (18 kDa molecular weight cut-off), which normally induces a complete run-down of the MgATP effect, does not alter the Pa stimulation of the exchanger, even after 6 h of continuous dialysis. We conclude that the ATP and Pa modulation of Na + -Ca 2+ exchange in an invertebrate nerve fibre are two genuinely different mechanisms, which affect the carrier properties in very different ways. An interesting similarity between ATP and Pa is that a phosphorylation-dephosphorylation process seems to be a common feature of these two regulation modes.