CLONING, EXPRESSION, AND CHARACTERIZATION OF THE SQUID NA-CA2+ EXCHANGER (NCX-SQ1)()

We have cloned the squid neuronal Na+-Ca2+ exchanger, NCX-SQ1, express ed it in Xenopus oocytes,and characterized its regulatory and ion tran sport properties in giant excised membrane patches. The squid exchange r shows 58% identity with the canine Na+-Ca2+ exchanger (NCX1.1). Regi ons determined to be of functional importance in NCX1 are well conserv ed. Unique among exchanger sequences to date, NCX-SQ1 has a potential protein kinase C phosphorylation site (threonine 184) between transmem brane segments 3 and 4 and a tyrosine kinase site in the Ca2+ binding region (tyrosine 462). There is a deletion of 47 amino acids in the la rge intracellular loop of NCX-SQ1 in comparison with NCX1. Similar to NCX1, expression of NCX-SQ1 in Xenopus oocytes induced cytoplasmic Na-dependent Ca-45(2+) uptake; the uptake was inhibited by injection of Ca2+ chelators. In giant excised membrane patches, the NCX-SQ1 outward exchange current showed Na+-dependent inactivation, secondary activat ion by cytoplasmic Ca2+, and activation by chymotrypsin. The NCX-SQ1 e xchange current was strongly stimulated by both ATP and the ATP-thioes ter, ATP gamma S, in the presence of F- (0.2 mM) and vanadate (50 mu M ), and both effects reversed on application of a phosphatidylinositol- 4,5'-bisphosphate antibody. NCX1 current was stimulated by ATP, but no t by ATP gamma S. Like NCX1 current, NCX-SQ1 current was strongly stim ulated by phosphatidylinositol-4',5'-bisphosphate liposomes. In contra st to results in squid axon, NCX-SQ1 was not stimulated by phosphoargi nine (5-10 mM).After chymotrypsin treatment, both the outward and inwa rd NCX-SQ1 exchange currents were more strongly voltage dependent than NCX1 currents. Ion concentration jump experiments were performed to e stimate the relative electrogenicity of Na+ and Ca2+ transport reactio ns. Outward current transients associated with Na+ extrusion were much smaller for NCX-SQ1 than NCX1, and inward current transients associat ed with Ca2+ extrusion were much larger. For NCX-SQ1, charge movements of Ca2+ transport could be defined in voltage jump experiments with a low cytoplasmic Ca2+ (2 mu M) in the presence of high extracellular C a2+ (4 mM). The rates of charge movements showed ''U''-shaped dependen ce 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 m oves into the membrane field in NCX-SQ1 than in NCX1 when ions are occ luded into binding sites.