Beta-Adrenergic Regulation Of A Novel Isoform Of Ncx: Sequence And Expression Of Shark Heart Ncx In Human Kidney Cells

Janowski E, Day R, Kraev A, Roder JC, Cleemann L, Morad M. beta-Adrenergic regulation of a novel isoform of NCX: sequence and expression of shark heart NCX in human kidney cells. Am J Physiol Heart Circ Physiol 296: H1994-H2006, 2009. First published April 24, 2009; doi: 10.1152/ajpheart.00038.2009.-The function, regulation, and molecular structure of the cardiac Na+/Ca2+ exchangers (NCXs) vary significantly among vertebrates. We previously reported that beta-adrenergic suppression of amphibian cardiac NCX1.1 is associated with specific molecular motifs. Here we investigated the bimodal, cAMP-dependent regulation of spiny dogfish shark (Squalus acanthias) cardiac NCX, exploring the effects of molecular structure, host cell environment, and ionic milieu. The shark cardiac NCX sequence (GenBank accession no. DQ 068478) revealed two novel proline/alanine-rich amino acid insertions. Wild-type and mutant shark NCXs were cloned and expressed in mammalian cells (HEK-293 and FlpIn-293), where their activities were measured as Ni2+-sensitive Ca2+ fluxes (fluo 4) and membrane (Na2+/Ca2+ exchange) currents evoked by changes in extracellular Na+ concentration and/or membrane potential. Regardless of Ca2+ buffering, beta-adrenergic stimulation of cloned wild-type shark NCX consistently produced bimodal regulation (defined as differential regulation of Ca2+-efflux and -influx pathways), with suppression of the Ca2+-influx mode and either no change or enhancement of the Ca2+-efflux mode, closely resembling results from parallel experiments with native shark cardiomyocytes. In contrast, mutant shark NCX, with deletion of the novel region 2 insertion, produced equal suppression of the inward and outward currents and Ca2+ fluxes, thereby abolishing the bimodal nature of the regulation. Control experiments with nontransfected and dog cardiac NCX-expressing cells showed no cAMP regulation. We conclude that bimodal beta-adrenergic regulation is retained in cloned shark NCX and is dependent on the shark's unique molecular motifs.