Bagriantsev SN , Chatelain FC , Clark KA , Alagem N , Reuveny E , Minor DL .

R01 MH093603 NIMH NIH HHS, R01-MH093603 NIMH NIH HHS, R01-NS049272 NINDS NIH HHS

	Figure 1. (A) Schematic showing the library display strategy used for identifying Kir3.2 activators. (left) Representations of the random peptide, random peptide (green) constrained by pZ (ribbons), and a pZ surface display library (magenta positions on blue ribbon diagram) were linked to the N- or C-terminal cytoplasmic ends of Kir3.2. (right) Cartoon showing a single Kir3.2 subunit bearing a representative of the N- or C-terminal pZ surface library. (B) Potassium transport complementation assay shows the ability of N5-Kir3.2 to restore growth of the trk1Δtrk2Δ yeast on limiting potassium conditions (1 mM KCl). (C) Sequence alignment of pZ and N5. pZ helical segments are shown in large type. Positions randomized in the pZ surface library and the N5 residues at the randomized positions are shown in blue and orange for Helix 1 and Helix 2, respectively. (D) Exemplar two-electrode voltage clamp recordings from Xenopus oocytes injected with 3 ng of cRNA for Kir3.2, N5-Kir3.2, and pZ-Kir3.2. Lower right panel shows voltage protocol used for recordings. Currents were evoked by 1 s long step protocol from −110 to 40 mV, in 10 mV increments from a holding potential of 0 mV in 90K. (E) Exemplar current–voltage plots for the indicated constructs recorded in 90K with or without 5 mM BaCl2. (F) Quantification the effects of pZ and N5 on activity of Kir3.2, Kir3.1, Kv7.2, and TRPM8 currents. Kir3.2 were evoked as in (D) and measured at −80 mV. Kv7.2 currents were evoked by 2.5 s long step protocol from −110 to 40 mV in 10 mV increments from a holding potential of −80 mV and measured at +30 mV at 2.4 s. TRPM8 currents were evoked in the presence of 250 μM menthol by a 900 ms long ramp from −110 to 40 mV, from a holding potential of −60 mV, and measured at 30 mV. Data shown as mean ± standard error of the mean (SEM) n ≥ 6). “nd” indicates not determined.
	Figure 2. Exemplar recordings (A) and quantification (B) of the effect of 3 μM carbachol on N5-Kir3.2 activity in Xenopus oocytes coinjected with 5 ng of mAChR. Data in (B) are mean ± SD (n ≥ 6). (C) Quantification of surface expression of HA-tagged Kir3.2 and N5-Kir3.2 as a function of injected cRNA. Data are mean ± SEM (n ≥ 6). Inset depicts cartoons of a single subunit of the HA-tagged Kir3.2 and N5-Kir3.2 constructs. (D) Immunoblot analysis of total lysates from oocytes injected with different amounts of cRNA. (E) Quantification of surface expression of N5-Kir3.2-HA in the presence of 15 μM brefeldin A (BFA). Surface fluorescence (SF) measurements (mean ± SEM, n ≥ 6) were normalized to the initial fluorescence values and fitted to the single exponential decay equation: SF = (1 – SFplateau) exp(−Kt) + SFplateau, where t is the time in h, SFplateau is the minimal fluorescence, and K is the decay constant.
	Figure 3. (A) Coomassie-stained SDS-PAGE of purified recombinant pZ and N5. (B) Comparison of pZ and N5 CD spectra at 4 °C. (C) Thermal denaturation of pZ and N5, monitored by CD at 222 nm. CD data were measured in a buffer of 150 mM KCl, 4 mM β-mercaptoethanol, 10 mM phosphate, pH 7.4.
	Figure 4. Quantification of activity (A) , and surface expression (B) of N5-Kir3.2 mutants measured in Xenopus oocytes injected with 3 ng of cRNA of each construct. Currents were evoked by a 1 s long step protocol from −110 to 40 mV, in 10 mV increments, from a holding potential of 0 mV in 90K. Surface expression was measured by labeling of the surface of the oocytes with an α-HA antibody. Helix 1 and Helix 2 are indicated by blue and orange, respectively. Data are mean ± SEM (n ≥ 6). (C) Immunoblot analysis of total lysates from oocytes injected with 3 ng of cRNA of each of the indicated constructs.
	Figure 5. (A) Sequence alignment of wild-type and mutant N5 proteins. Quantification of activity (B) and surface expression (C) of indicated N5-Kir3.2 and mutants measured in Xenopus oocytes injected with 3 ng of cRNA of each construct. Currents were evoked by a 1 s long step protocol from −110 to 40 mV, in 10 mV increments, from a holding potential of 0 mV in 90K. Surface expression was measured by labeling of the surface of the oocytes with an α-HA antibody. Data are shown as mean ± SEM (n ≥ 6). (D) Immunoblot analysis of total lysates from oocytes injected with 3 ng of cRNA for the indicated constructs.
	Figure 6. (A) Exemplar current–voltage plots recorded in oocytes injected with 3 ng of cRNA for the indicated constructs. Currents were evoked by a 1 s long step protocol from −110 to 40 mV, in 10 mV increments, from a holding potential of 0 mV in 90K. (B) Quantification of activity and surface expression of N5mut-Kir3.2-HA channels in the oocytes injected with 3 ng of cRNA for each construct. Surface expression was measured by labeling of the surface of the oocytes with an α-HA antibody. Data are mean ± SEM (n ≥ 6) normalized to N5-Kir3.2-HA. (C) Immunoblot analysis of total lysates from oocytes injected with 3 ng of cRNA for the indicated constructs.
	Figure 7. (A) Exemplar two-electrode voltage clamp recordings from Xenopus oocytes injected with 3 ng of cRNA for HA-tagged Kir3.2 having N5 or pZ attached to the C-terminus (Kir3.2-HA-N5 and Kir3.2-HA-pZ, respectively). Currents were evoked by a 1 s long step protocol from −100 to 50 mV, in 10 mV increments from a holding potential of 0 mV in 90K. (B) Current–voltage plots recorded in 90K for Kir3.2-HA-pZ and for Kir3.2-HA-N5 with or without the addition of 5 mM BaCl2. (C) Quantification of activity and (D) surface expression of indicated Kir3.2-HA constructs measured in Xenopus oocytes injected with 3 ng of cRNA of each construct. Surface expression was measured by labeling of the surface of the oocytes with an anti-HA antibody. Data from at least two experiments were normalized to N5-Kir3.2-HA and shown as mean ± SEM (n = 8–25). (E) Immunoblot analysis of total lysates from oocytes injected with 3 ng of cRNA for the indicated constructs.

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