Roles of ³²⁹RKK³³¹ and the GR domain in modulation of P_o in Slo1 + β1 by PIP₂. (A) Schematic structural organization of a Slo1 subunit without the GR domain (Slo1ΔGR-Kv-minT). In Slo1ΔGR-Kv-minT, the polypeptide is truncated immediately C terminal to the sequence ³²⁹RKK³³¹. The distal red segment represents the amino acids added by Budelli et al. (2013) (GVKESLGGTDV). (B) Representative currents from Slo1ΔGR-Kv-minT + β1 before (blue) and after (red) the application of 10 µM PIP₂. (C) Fractional changes in peak outward currents at different voltages by 10 µM PIP₂ (red). The gray shaded area shows the mean ± SEM results from wild-type Slo1 + β1 for comparison. (D, F, and H) Illustrative currents through Slo1 R329A:K330A:K331A (D), Slo1 R329A:K330A:K331A + β1 (F), and Slo1 R329A:K330A:K331A + β4 (H) before (blue) and after (red) the application of 10 µM PIP₂. (E, G, and I) G-V curves of Slo1 R329A:K330A:K331A (“RKK mutant”), Slo1 R329A:K330A:K331A (“RKK mutant”) + β1, and Slo1 R329A:K330A:K331A (“RKK mutant”) + β4 before (blue circles) and after (red circles) the application of 10 µM PIP₂. For comparison, G-V curves from the respective wild-type Slo1 (E), Slo1 + β1 (G), and Slo1 + β4 (I) before (blue triangles) and after (red triangles) the application of 10 µM PIP₂ are also shown; n = 6–8. All results were obtained without Ca²⁺. Error bars represent mean ± SEM.