Cloning, Expression, and Characterization of the Squid Na(+)–Ca(2+) Exchanger (NCX-SQ1)

We have cloned the squid neuronal Na(+)–Ca(2+) exchanger, NCX-SQ1, expressed it in Xenopus oocytes, and characterized its regulatory and ion transport properties in giant excised membrane patches. The squid exchanger shows 58% identity with the canine Na(+)–Ca(2+) exchanger (NCX1.1). Regions determined to be of functional importance in NCX1 are well conserved. Unique among exchanger sequences to date, NCX-SQ1 has a potential protein kinase C phosphorylation site (threonine 184) between transmembrane segments 3 and 4 and a tyrosine kinase site in the Ca(2+) binding region (tyrosine 462). There is a deletion of 47 amino acids in the large intracellular loop of NCX-SQ1 in comparison with NCX1. Similar to NCX1, expression of NCX-SQ1 in Xenopus oocytes induced cytoplasmic Na(+)-dependent (45)Ca(2+) uptake; the uptake was inhibited by injection of Ca(2+) chelators. In giant excised membrane patches, the NCX-SQ1 outward exchange current showed Na(+)-dependent inactivation, secondary activation by cytoplasmic Ca(2+), and activation by chymotrypsin. The NCX-SQ1 exchange current was strongly stimulated by both ATP and the ATP-thioester, ATPγS, in the presence of F(−) (0.2 mM) and vanadate (50 μM), and both effects reversed on application of a phosphatidylinositol-4′,5′-bisphosphate antibody. NCX1 current was stimulated by ATP, but not by ATPγS. Like NCX1 current, NCX-SQ1 current was strongly stimulated by phosphatidylinositol-4′,5′-bisphosphate liposomes. In contrast to results in squid axon, NCX-SQ1 was not stimulated by phosphoarginine (5–10 mM). After chymotrypsin treatment, both the outward and inward NCX-SQ1 exchange currents were more strongly voltage dependent than NCX1 currents. Ion concentration jump experiments were performed to estimate the relative electrogenicity of Na(+) and Ca(2+) transport reactions. Outward current transients associated with Na(+) extrusion were much smaller for NCX-SQ1 than NCX1, and inward current transients associated with Ca(2+) extrusion were much larger. For NCX-SQ1, charge movements of Ca(2+) transport could be defined in voltage jump experiments with a low cytoplasmic Ca(2+) (2 μM) in the presence of high extracellular Ca(2+) (4 mM). The rates of charge movements showed “U”-shaped dependence on voltage, and the slopes of both charge–voltage and rate–voltage relations (1,600 s(−1) at 0 mV) indicated an apparent valency of −0.6 charges for the underlying reaction. Evidently, more negative charge moves into the membrane field in NCX-SQ1 than in NCX1 when ions are occluded into binding sites.