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Article

Zhaoping He^*, Qiusheng Tong, Beate D. Quednau^*, Kenneth D. Philipson^*, and Donald W. Hilgemann

From the ^* Departments of Physiology and Medicine and the Cardiovascular Research Laboratories, UCLA School of Medicine, Los Angeles, California 90095-1760; and Department of Physiology, University of Texas, Southwestern Medical Center at Dallas, Dallas, Texas 75235-9040

We have cloned the squid neuronal Na⁺–Ca²⁺ 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⁺–Ca²⁺ 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 Ca²⁺ 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 ⁴⁵Ca²⁺ 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 activation by cytoplasmic Ca²⁺, and activation by chymotrypsin. The NCX-SQ1 exchange current was strongly stimulated by both ATP and the ATP-thioester, ATPS, 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 ATPS. 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 Ca²⁺ transport reactions. Outward current transients associated with Na⁺ extrusion were much smaller for NCX-SQ1 than NCX1, and inward current transients associated 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 µM) in the presence of high extracellular Ca²⁺ (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.

Key Words: sodium–calcium exchange • charge movements • Xenopus oocytes • patch clamp • phosphatidylinositols

Abbreviations: MES, 2-(morpholino)ethanesulfonic acid; NMG, N-methyl-D-glucamine; nts, nucleotides; PIP₂, phosphatidylinositol-4',-5'-bisphosphate

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