Molecular determinants for verapamil block of Slo2.1 channels. (A) Amino acid sequence alignment of the pore region of Slo2.1 with other K⁺ channels. Sequence alignment begins with pore helix (PH) followed by the selectivity filter (SF) and S6 segment. Ser241, Thr242, and residues in S6 of human Slo2.1 that were mutated to Ala are underlined. Mutation of colored residues to Ala caused gain of function (green) or loss of function (blue). P475 in Shaker and E92 in MthK channel are colored red. (B) Bar graph summarizing relative block of I_Slo2.1 (I_verapamil/I_control) by 15 µM verapamil for WT channels and channels with indicated point mutation. The broken horizontal line indicates I_verapamil/I_control value for WT channels. Currents were activated by 1 mM NFA and elicited with 300-ms pulses to 0 mV. Error bars indicate mean ± SEM. (C) Homology model of Slo2.1 pore domain viewed from cytoplasmic side and showing side chains of Ser241 (yellow), Val268 (magenta), and Phe274 (cyan). (D) Concentration-dependent block of WT Slo2.1 channel currents by verapamil. I_Slo2.1 activated by 1 mM NFA in the absence (control) and presence of indicated [verapamil]. Pulses were applied to 0 mV for 300 ms. (E) [Verapamil]–response curve for WT and mutant Slo2.1 channels. The IC₅₀ was 14.5 ± 0.6 µM (n_H = 1.5 ± 0.1; n = 7) for WT channels, 58.7 ± 9.4 µM (n_H = 1.1 ± 0.2, n = 6) for F274T channels activated by 1 mM NFA (red circles), 53.0 ± 2.4 µM (n_H = 1.0 ± 0.06, n = 4) for constitutively active F274T channels (blue triangles), and 4.6 ± 2.3 µM (n_H = 1.4 ± 0.5, n = 4) for V268A channels (teal circles). Error bars indicate mean ± SEM.