Fig. 4
Design of minimal Glu-gated viral potassium channels. a Schematic drawings and functional expression of the deletion and mutant constructs used. Cartoons depicting the NTD- and the M4 truncated versions and the cysteines mutated (indicated by red stars); Inset: Point mutations in the LBD (N407C) and TM1 segment (V152C) thought to form a disulfide-bridge are highlighted in red based on our homology model against 3KG2. Numbers correspond to amino acid positions in the mature protein. Imax currents of each mutant, with and without DTT in the case of the cysteine double mutant, are shown (p = 0.8; one-way ANOVA with turkey correction; n = 4–10). b Overlay of representative whole cell current traces of Glu-gated GluATCV*, GluATCV*ΔNTD ΔM4, and GluA1. The respective traces in response to application of 100 µM glutamate (Glu) are shown in magenta, black, and orange, respectively. Note that the overall shape of the glutamate-induced whole cell currents is similar between the constructs. c Glu dose–response curves of GluATCV* (black circles), the deletion mutant GluATCV*ΔNTD ΔM4 (magenta squares), and the double mutant GluATCV*ΔNTD ΔM4 V152/N402C (green triangles) without DTT (open symbols) and in the presence of 5 mM DTT (closed symbols). EC50 GluATCV* 5.8 ± 1.1 µM (−DTT) and 5.1 ± 1.1 µM (+DTT); GluATCV*ΔNTD ΔM4 20.8 ± 2.6 µM (−DTT) and 22.6 ± 2.9 µM (+DTT); GluATCV*ΔNTD ΔM4 V152/N402C 5.9 ± 0.4 µM (−DTT) and 10.5 ± 0.7 µM (+ DTT). Note that, in contrast to GluATCV* and GluATCV*ΔNTD ΔM4, only the GluATCV*ΔNTD ΔM4 V152/N402C shows a significant changed EC50 value in the presence of DTT (green closed triangles) indicative of an intrasubunit disulfide link between LBD and TM1
