β-adrenergic Regulation of a Novel Isoform of NCX: Sequence & Expression of Shark Heart NCX in Human Kidney Cells

Overexpression of the Na+/Ca2+ exchanger (NCX) in mammals is associated with arrhythmias in cardiac hypertrophy and failure. The function and regulation of NCX genes varies significantly among vertebrates likely reflecting differences in molecular structure. We have previously reported that β-adrenergic suppression of the Ca2+-efflux and -influx modes of amphibian cardiac NCX1.1 is associated with specific molecular motifs. In contrast, cardiac NCX of shark (Squalus acanthias) shows ‘bimodal’ adrenergic regulation with preferential suppression of the Ca2+-influx mode (Woo and Morad, PNAS 98:2023, 2001); and its sequence (DQ 068478) reveals two novel proline/alanine-rich AA-insertions. Here we examined the effects of deleting the longer of these inserts.Shark and mutant shark cardiac NCX were expressed in mammalian cells (HEK 293 and FlpIn 293), and their activity was measured as Ni2+-sensitive Ca2+-fluxes (Fluo-4) and membrane currents (INaCa) by changing [Na+]o and/or membrane potential (Vm). Bimodal regulation, defined as differential regulation of Ca2+-efflux and influx pathways with a strong suppression of its Ca2+-influx mode and no change, or enhancement, of the Ca2+-efflux mode, persisted in the shark NCX regardless of Ca2+ buffering, closely resembling the β-adrenergic regulation of native shark cardiomyocytes. In contrast, β-adrenergic stimulation of the shark mutant NCX produced an equal suppression of the inward and outward currents as well as the Ca2+ fluxes (as found with frog NCX), thereby abolishing the bimodal nature of the regulation. Control experiments were carried out with untransfected and dog cardiac NCX expressing cells.We conclude that shark NCX is physiologically functional in mammalian cells, retaining the essentials of its bimodal β-adrenergic regulation. In addition, the deleted shark-specific insert was found to affect the modality of cAMP-dependent regulation, possibly because it provides essential intramolecular flexibility and/or binding sites.