Transportadores concentrativos de nucleósidos CNT1 y CNT3 humanosestudio funcional y caracterización de polimorfismos mediante técnicas electrofisiológicas

Resumen

The human SLC28 family of integral membrane concentrative nucleoside transporter proteins has three members, hCNT1, hCNT2 and hCNT3. Na+-coupled hCNT1 and hCNT2 transport pyrimidine and purine nucleosides, respectively, whereas hCNT3 mediates transport of both pyrimidine and purine nucleosides utilizing Na+ or H+ electrochemical gradients. We expressed hCNT1 and hCNT3 in Xenopus laevis oocytes and examined its steady-state and presteady-state kinetics using electrophysiological methods. For hCNT3, the maximal current for uridine and cations increased with hyperpolarizing voltages with no evidence of saturation, suggesting that there is at least one rate-limiting voltage-dependent step in the transport cycle. When transport was activated by Na+, the apparent uridine affinity decreased with negative potentials, whereas in H+ decreased as membrane potential become more positive. In H+, inosine was transported by hCNT3 with the same affinity than uridine but at 30% of its transport rate. Our experiments indicate that the rate-limiting step for inosine transport is nucleoside translocation, whereas for uridine it is reorientation of the empty transporter in the membrane. In response to step voltage changes, hCNT3 exhibited current transients. Maximal charge was conserved upon progressive removal of cations, whereas the voltage midpoint (V0.5) shifted to more hyperpolarized potentials. Therefore, charge movements in hCNT3 are primarily due to the conformational changes of the empty transporter within the membrane electric field. The hCNT3 polymorphism Cys602Arg, showed a decreased in Na+ affinity and a change in substrate selectivity. In H+, both cation and uridine affinity was increased. For hCNT1, the rare variant Ser546Pro did not transport nucleosides but showed uncoupled Na+ transport.