Pharmacological properties of naturally occurring variants of the human norepinephrine transporter

The human norepinephrine transporter (hNET) gene has five sequence polymorp hisms that predict amino acid substitutions in the transporter protein: Val (69)Ile, Thr(99)Ile, Val(245)Ile, Val(449)Ile, and Gly(478)Ser. In order to functionally characterize the naturally occurring transporter variants, we used site-directed mutagenesis to establish the hNET variants and compared some basic pharmacological properties (uptake of norepinephrine and its in hibition by the tricyclic antidepressant desipramine) in COS-7 cells transi ently expressing variant hNETs and wild-type hNET. None of the hNET variant s displayed changes in the potency (Ki) of desipramine for inhibition of no repinephrine uptake. Furthermore, variants Val(69)Ile, Thr(99)Ile, Val(245) Ile, and Val(449)Ile did not affect kinetic constants (K-m, V-max) of norep inephrine uptake, However, COS-7 cells expressing the hNET variant Gly(478) Ser displayed an approximately four-fold increase in the K-m for norepineph rine, while the V-max was unaffected, The increase in the K-m, which is equ ivalent to a four-fold reduction in the affinity of the variant hNET for it s natural substrate norepinephrine, indicates that the glycine in position 478 is part of a substrate recognition domain, The reduced clearance of rel eased norepinephrine by reuptake through the Gly(478)Ser variant might caus e an increase in the synaptic and the circulating concentration of norepine phrine. Elevated norepinephrine concentrations have been associated with hu man diseases and it will be interesting to explore a possible contribution by the Gly(478)Ser variant to certain desease states, Pharmacogenetics 10:3 97-405 (C) 2000 Lippincott Williams & Wilkins.