title: Cloning, expression, and characterization of the squid Na+-Ca2+ exchanger (NCX-SQ1).
abstract: 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, ATP gamma S, in the presence of F- (0.2 mM) and vanadate (50 microM), and both effects reversed on application of a phosphatidylinositol-4',5'-bisphosphate antibody. NCX1 current was stimulated by ATP, but not by ATP gamma S. 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 microM) 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.
authors.affiliation: Department of Physiology, Cardiovascular Research Laboratories, UCLA School of Medicine, Los Angeles, California 90095-1760, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: He
authors.forename: Q
authors.initials: Q
authors.lastname: Tong
authors.forename: B D
authors.initials: BD
authors.lastname: Quednau
authors.forename: K D
authors.initials: KD
authors.lastname: Philipson
authors.forename: D W
authors.initials: DW
authors.lastname: Hilgemann
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular; D049832:Decapodiformes / Q000378:metabolism*; D004558:Electric Stimulation; D004594:Electrophysiology; D008564:Membrane Potentials / Q000502:physiology; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D019831:Sodium-Calcium Exchanger / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D014982:Xenopus laevis
chemical_list: D019831:Sodium-Calcium Exchanger