A novel 13 kDa cytoplasmic soluble protein is required for the nucleotide (MgATP) modulation of the Na/Ca exchange in squid nerve fibers

Abstract

The Na/Ca exchange is a highly regulated transport mechanism in which MgATP, a powerful modulatory intracellular substrate, has important implications for its function. As occurs with some preparations, in squid axons, nucleotide regulation is lost after membrane vesicle isolation. This has been a significant obstacle in the biochemical characterization of the MgATP effect. An important clue in solving this long-standing puzzle is presented in this work by showing that prolonged intracellular dialysis of squid axons produces a complete run down of the MgATP effect. Here we report that a soluble cytoplasmic factor isolated from fresh squid axoplasm and brain reconstitutes the MgATP stimulation of the Na-gradient-dependent 45Ca uptake in squid optic nerve membrane vesicles. Partial purification of this factor uncovers the presence of a novel 13 kDa soluble cytoplasmic protein (SCPr) which, when microinjected in ATP de-regulated dialyzed squid axons, completely restores the MgATP stimulation of Na(o)-dependent Ca efflux. We propose that in the squid preparation this SCPr constitutes the link between the nucleotide and target effector: the Na/Ca exchanger itself, or other plasma membrane structures which may secondarily interact with the exchanger.

Sodium/Calcium Exchanger: Influence of Metabolic Regulation on Ion Carrier Interactions

“…This finding led to the idea that a low-molecular-weight cytosolic compound lost during prolonged dialysis was essential, and efforts were started to isolate it. Initially, the starting material was axoplasm extruded from squid giant axons (23); identical results were later obtained by using optic ganglia with the advantage that more material was obtained (94). Figure 25, top, shows the present procedure used for the partial purification of this soluble cytosolic regulatory protein (SCRP).…”

“…This was circumvented by the development of the inside-out excised giant patch (25-30 m diameter) in cardiac cells (120,123), which allows the study of kinetics and regulatory processes related to the Na ϩ /Ca 2ϩ exchanger function. One main disadvantage is that during the formation of the giant patch, cell components (among them soluble proteins that may be involved in the Na ϩ / Ca 2ϩ exchange regulation) are eliminated (23,94). Finally, substantial progress has been made in the molecular biology of the Na ϩ /Ca 2ϩ exchanger (204).…”

“…With the use of nerve vesicles, the appearance of MgATP stimulation was observed only in the number 65 elution fraction that corresponded to a molecular mass of ϳ13 kDa. When that fraction was injected into axons lacking the MgATP effect due to prolonged dialysis, the upregulation by the nucleotide was restored (23,94); other fractions were not effective. Similar results were obtained with MgATP␥S (Berberiá n, Di-METABOLIC AND IONIC REGULATION OF NA ϩ /CA 2ϩ EXCHANGE Polo, and Beaugé , unpublished data) but not with the nonhydrolyzable ATP analogs AMP-PCP and AMP-PNP.…”

Cardiac Na + -Ca 2+ Exchange

“…In this context, it is important to note that a novel 13-kDa cytosolic protein recently isolated from the axoplasm and brain of squid has been shown to be involved in ATP-dependent regulation of NCX-SQ1. 52 Protein kinase A activation by forskolin and/or other reagents has been reported to stimulate activities of cardiac NCX1 expressed in BHK cells 50 and Xenopus oocytes, 24 although the same clone expressed in CCL39 cells is not affected by 8-bromo-cAMP or 8-bromo-cGMP. 48 In frog heart, however, ␤-receptor-dependent or cAMP-dependent stimulation results in the inhibition of NCX activity, in which a novel 9 -amino acid exon of the frog exchanger seems to play a role.…”

Functional comparison of the reverse mode of Na+/Ca2+ exchangers NCX1.1 and NCX1.5 expressed in CHO cells

“…In addition, studies testing the regulation of NCX1.5 by PKA and PKC were reported rarely. Some authors reported that a phosphorylation step could accelerate the transporting function of the Na + /Ca 2+ exchanger [12][13][14] , but others failed to observe the same effect [15,16] .…”