Identification of Cys(140) in helix 4 as an exofacial cysteine residue within the substrate-translocation channel of rat equilibrative nitrobenzylthioinosine (NBMPR)-insensitive nucleoside transporter rENT2

The human and rat equilibrative nucleoside transporter proteins hENT1, rENT 1, hENT2 and rENT2 belong to a family of integral membrane proteins with 11 potential transmembrane segments (TMs), and are distinguished functionally by differences in transport of nucleobases and sensitivity to inhibition b y nitrobenzylthioinosine (NBMPR) and vasoactive drugs, In the present study , we have produced recombinant hENT1, rENT1, hENT2 and rENT2 in Xenopus ooc ytes and investigated uridine transport following exposure to the impermean t thiol-reactive reagent p-chloromercuriphenyl sulphonate (PCMBS). PCMBS ca used reversible inhibition of uridine influx by rENT2, but had no effect on hENT1, hENT2 or rENT1. This difference correlated with the presence in rEN T2 of a unique Cys residue (Cys(140)) in the outer half of TM4 that was abs ent from the other ENTs. Mutations of Cys(140) to Ser produced a functional protein (rENT2/C140S) that was insensitive to inhibition by PCMBS, identif ying Cys(140) as the exofacial Cys residue in rENTZ responsible for PCMBS i nhibition. Uridine protected wild-type rENT2 against PCMBS inhibition, sugg esting that Cys(140) in TM4 lies within or is closely adjacent to the subst rate-translocation channel of the transporter. TM4 has been shown previousl y to be within a structural domain (TMs 3-6) responsible for interactions w ith NBMPR, vasoactive drugs and nucleobases.