Peak and steady SERT currents induced by rapid application of 5-HT. (A) Application of 5-HT to a SERT-expressing HEK293 cell perfused with KCl in the absence of internal Na⁺. The peak current was induced on application of 5-HT and the steady current continued as long as 5-HT was present. (B–E) Current traces obtained under the conditions shown, with the partial reaction cycle proposed to be associated with those traces. (B) In the absence of internal alkali cations, a strong peak current was observed with no steady current. The dashed box shows the steps responsible for the peak current. (C) Addition of high internal Na⁺ suppressed the peak current. The peak current was smaller than in A, because at high intracellular Na⁺, the Na2 site was mostly occupied, and thus, there was little net flux of Na⁺ into the cytosol. (D and E) Addition of internal K⁺ at intermediate (D) or high (E) concentrations progressively increased the steady current and decreased the peak current. The dashed box shows a hypothetical intermediate responsible for the steady current. (F) Two-pulse protocol for measuring recovery of the outward-facing conformation of SERT. Traces from six trials are shown, with arrows indicating additions of extracellular 5-HT. Red boxes indicate when the second pulse was applied. The current recovered with a monoexponential time course. The rate of current recovery is a measure of the turnover rate.