Yau MC , Kim RY , Wang CK , Li J , Ammar T , Yang RY , Pless SA , Kurata HT .

MOP 142482 CIHR

	Figure 1. A biphasic conductance–voltage relationship emerges at intermediate retigabine concentrations. (A) Two-electrode voltage-clamp recordings from X. laevis oocytes expressing KCNQ3* after exposure to 0, 3, and 300 µM retigabine (RTG). Oocytes were held at −80 mV and depolarized for 2 s to voltages between −140 mV and +20 mV (in 5-mV steps), followed by repolarization to a −20 mV test potential. Highlighted in the insets are the tail currents elicited at −20 mV in 3 µM retigabine, illustrating the clustering of tail current amplitude at intermediate voltages and the saturating effect in 300 µM retigabine. (B) Conductance–voltage relationships were determined in different retigabine concentrations using normalized tail current amplitudes immediately after the voltage step to −20 mV. Gray lines are fits (sum of two Boltzmann equations) of data from individual oocytes in 3 µM retigabine, and the circles represent averages. Fully activated currents near the reversal potential were measured by stepping through a range of voltages after an activating prepulse to 20 mV (inset). Peak tail current magnitude was normalized to currents elicited at 20 mV. Retigabine does not appear to alter the reversal potential of KCNQ3-mediated currents.
	Figure 2. Conductance–voltage relationships for KCNQ3* in different retigabine concentrations. (A) Conductance–voltage relationships were determined over a range of retigabine (RTG) concentrations using normalized tail current amplitudes from the same voltage step protocol as Fig. 1 A. Conductance–voltage relationships are fit with the sum of two Boltzmann equations (fit parameters are summarized in Table 1). (B and C) The concentration response to retigabine was determined using the fraction of the shifted Boltzmann component (B) or the normalized current (−60 mV; C) at different retigabine concentrations and fit with a Hill equation. Data presented are mean ± SEM.
	Figure 3. Generation of KCNQ3* concatemers with variable stoichiometry of retigabine-sensitive subunits. (A) Western blots obtained from HEK293 cells expressing tetrameric constructs show predominant expression of tetrameric KCNQ3 protein, and the absence of monomeric subunits, in comparison to cells transfected with monomeric KCNQ3* control. (B and C) Representative activation and deactivation kinetics and summarized conductance–voltage relationships for all concatenated KCNQ3* channel constructs (W represents a native Trp at position 265, and F represents a Trp265Phe mutant subunits). No significant differences in V1/2 or slope factor were detected in comparisons between the different concatenated channel types. Data points are mean ± SEM.
	Figure 4. WWWW concatenated channels exhibit similar retigabine responses as homomeric KCNQ3*. (A) Conductance–voltage relationships were collected in multiple retigabine (RTG) concentrations using the same voltage protocol as Fig. 1. Overlaid blue curves are fits described in Fig. 2 and Table 1 for KCNQ3[A315T] channels (sum of two Boltzmann equations). Data are mean ± SEM. (B) Exemplar currents collected in 3 µM retigabine, illustrating the progression of tail currents that underlie the biphasic conductance–voltage relationship.
	Figure 5. A single retigabine-sensitive subunit encodes a large retigabine effect. (A–D) Summary conductance–voltage relationships from WWWF (A), WWFF (B), WFFF (C), and FFFF (D) concatenated constructs, expressed in X. laevis oocytes and incubated in various retigabine (RTG) concentrations. (E) Summary data of the concentration–response of retigabine-mediated channel activation at −60 mV. (F) Maximal RTG-induced shift in V1/2 for different constructs. See also Table 2 (data for FFFF are not shown). Data are mean ± SEM.
	Figure 6. Effects of retigabine on the activation and deactivation kinetics of KCNQ3* tetramers. (A) Sample activation (left, 0-mV pulse) and deactivation (right, −140-mV pulse) traces obtained in the presence (red) or absence (black) of a saturating concentration of retigabine (RTG). (B and C) Summary of kinetic data for all concatenated KCNQ3* constructs (logτact, logτdeact). Notice that kinetic effects of retigabine are apparent even in concatenated channels with a single retigabine-sensitive subunit. Data are mean ± SEM.
	Figure 7. Distinct current relaxation in WWWW and WFFF after rapid retigabine wash off. (A and B) WWWW and WFFF concatemers were expressed in HEK293 cells, and currents were measured by patch clamp. Cells were held at various voltages, and a rapid solution-switching system was used to apply retigabine, followed by a rapid wash off into control conditions. Exemplar traces illustrating current relaxation after retigabine wash off are presented for WWWW and WFFF at −60 mV (A) and −20 mV (B), as indicated.
	Figure 8. Supershifted retigabine-binding site mutants highlight an all-or-none shift. (A) Molecular model highlighting the positioning of L314 and L272 in close proximity to W265 in the putative retigabine (RTG)–binding site (residues highlighted in yellow). (B) Concentration responses of KCNQ3* and binding site mutants measured using the normalized conductance at −60 mV. (C and D) Summary conductance–voltage relationships obtained from KCNQ3*[L314A] and KCNQ3*[L272I] channels in different retigabine concentrations using the same protocol as Figure 1. Inset panels display sample tail current recordings in 30 µM retigabine, clearly highlighting clustered tail currents after prepulses to intermediate voltages.

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