Permeation and gating residues in serotonin transporter

The third transmembrane domain (TM3) of serotonin transporter (SERT) contai ns two isoleucine residues previously proposed to be involved in binding an d transport: of serotonin. When Ile-172 was replaced with cysteine, SERT be came sensitive to inactivation by externally added [2-(trimethylammonium)et hyl]methanethio-sulfonate (MTSET). The disulfide product of this inactivati on was not sensitive to reduction by externally added sulfhydryl compounds, but apparently reacted with intracellular reducing agents to spontaneously regenerate active SERT. The apparent accessibility of this residue to both external and cytoplasmic reagents is consistent with its localization near a serotonin binding site that is alternately exposed to both internal and external media. In another SERT mutant, I179C, transport also was inactivat ed by MTSET but substrate binding was resistant. External substrate bound t o the inactivated I179C and enhanced its reactivation by free thiols, In no repinephrine transporter (NET), cysteine replacement of Ile-155 (correspond ing to SERT Ile-179) also rendered the transporter sensitive to MTSET inact ivation. In NET I155C, cocaine enhanced this inactivation, and the substrat e, dopamine, apparently protected against inactivation. The characteristics of this protection suggest that dopamine was transported, converting NET t o a form in which Ile-155 was occluded, The results support the proposal th at TM3 of SERT and NET constitute part of the substrate permeation pathway, and that Ile-172 in SERT resides close to the substrate binding site. They also suggest that Ile-179 in SERT (and Ile-155 in NET) is in a conformatio nally sensitive part of TM3, which may act as part of an external gate.