Novel regulation of equlibrative nucleoside transporter 1 (ENT1) by receptor-stimulated Ca2+-dependent calmodulin binding

Equilibrative nucleoside transporters (ENTs) facilitate the flux of nucleosides, such as adenosine, and nucleoside analog (NA) drugs across cell membranes. A correlation between adenosine flux and calcium-dependent signaling has been previously reported; however, the mechanistic basis of these observations is not known. Here we report the identification of the calcium signaling transducer calmodulin (CaM) as an ENT1-interacting protein, via a conserved classic 1-5-10 motif in ENT1. Calcium-dependent human ENT1-CaM protein interactions were confirmed in human cell lines (HEK293, RT4, U-87 MG) using biochemical assays (HEK293) and the functional assays (HEK293, RT4), which confirmed modified nucleoside uptake that occurred in the presence of pharmacological manipulations of calcium levels and CaM function. Nucleoside and NA drug uptake was significantly decreased (similar to 12% and similar to 39%, respectively) by chelating calcium (EGTA, 50 mu M; BAPTA-AM, 25 mu M), whereas increasing intracellular calcium (thapsigargin, 1.5 mu M) led to increased nucleoside uptake (similar to 26%). Activation of N-methyl-D-aspartate (NMDA) receptors (in U-87 MG) by glutamate (1 mM) and glycine (100 mu M) significantly increased nucleoside uptake (similar to 38%) except in the presence of the NMDA receptor antagonist, MK-801 (50 mu M), or CaM antagonist, W7 (50 mu M). These data support the existence of a previously unidentified novel receptor-dependent regulatory mechanism, whereby intracellular calcium modulates nucleoside and NA drug uptake via CaM-dependent interaction of ENT1. These findings suggest that ENT1 is regulated via receptor-dependent calcium-linked pathways resulting in an alteration of purine flux, which may modulate purinergic signaling and influence NA drug efficacy.