Corbin-Leftwich A , Mossadeq SM , Ha J , Ruchala I , Le AH , Villalba-Galea CA .

KL2 TR000057 NCATS NIH HHS , KL2TR000057 NCATS NIH HHS , UL1 TR000058 NCATS NIH HHS , UL1TR000058 NCATS NIH HHS

	Figure 1. K+ deactivation rate decreases as a function of activation. (A) K+ currents recorded from Xenopus oocytes coexpressing human KV7.2 and KV7.3 channels using the cut-open voltage-clamp technique. Test pulses ranging from −120 mV to +60 mV were applied from an HP of −90 mV. Deactivation of the current was driven at −105 mV (arrow). (B) Normalized deactivating currents from activating pulses from −30 to +60 mV. Deactivation slowed down after higher activating potentials. (C) Deactivation time constant (τDEACT) versus test pulse potential (τDEACT–VPULSE) plot. Error bars represent standard deviation. (D) Kinetics scheme describing alternative pathways for the deactivation of the heteromeric KV7.2/KV7.3 channel. Voltage-dependent transitions are noted with V.
	Figure 2. Effect of Retigabine on the activity of the heteromeric KV7.2/KV7.3 channel. (A) K+ currents recorded in the absence or presence of 1–5-µM Retigabine using the same protocol shown in Fig. 1. (B) Average normalized K+ current amplitude measured at the beginning of the deactivating currents (ITAIL; arrows in A). Normalized ITAIL versus test pulse potential (ITAIL–VPULSE) plots incrementally shift toward negative potentials as the concentration of Retigabine (RTG) was increased. Individual ITAIL–VPULSE plots were fitted to a double Boltzmann distribution, and the weighted average half-maximum potential (weighted V1/2) was plotted as a function of the concentration of Retigabine (inset). Although the weighted V1/2 values were not statistically different, this parameter changed to more negative potentials as a function of the concentration of Retigabine in all our recordings. Error bars represent standard deviation. n = 5–8.
	Figure 3. Deactivation time constant increase as a function of the duration of the activating pulse. (A and B) K+ currents were activated by 40-mV pulses with a duration varying from 10 to 4,175 ms in the absence (A) and presence (B) of 1-µM Retigabine. The duration of the pulse increased 1.3-fold for each trace. After activation, the K+ current was deactivated at −90 mV. (C) τDEACT–tPULSE plot shows that τDEACT increased as the activating pulse was longer (black squares). In the presence of Retigabine, τDEACT increased further. (D) The same plot as in C, but the tPULSE is displayed in a logarithmic scale to highlight the τDEACT–tPULSE relationship for shorter activating pulses and showing that τDEACT was unaltered by Retigabine for pulses shorter than 500 ms. Error bars represent standard deviation. Control, n = 7; Retigabine, n = 6.
	Figure 4. Deactivation from the depolarized and resting potential open states. (A) Deactivation at −90 mV of the K+ current elicited by a 100-ms depolarization to 40 mV in the absence (black trace) and presence of 1-µM Retigabine (red trace). (B) Deactivation at −90 mV of the K+ current elicited by holding the membrane potential at −50 mV also in the absence (black trace) and presence of 1-µM Retigabine (red trace). (C–-F) Deactivation time constant (τDEACT) versus deactivating potential (τDEACT–VDEACT) plot of channels activated by a 100-ms depolarization to 40 mV (C) or by holding the membrane potential at −60 mV, −50 mV, and −40 mV (D, E, and F, respectively) in the absence (black squares) and presence of 1-µM Retigabine (RTG; red circles). Error bars represent standard deviation. t test; n = 5–8. *, P < 0.05, t test.
	Figure 5. The resting potential, but not the threshold potential for triggering AP, was affected by Retigabine. (A) APs were recorded from Xenopus oocytes coexpressing both the α and β subunits of NaV1.4, ShakerIR, KV7.2, and KV7.3. The APs were recorded using the loose two-electrode voltage-clamp technique (see Materials and methods for details) in the absence (black trace) or presence of 1-µM (red trace) or 10-µM (green trace) Retigabine. The gray trace shows the response of VM when a subthreshold was applied. The arrow indicates the moment at which AP threshold (VTHR) is reached. (B) Effect of Retigabine (RTG) on the resting membrane potential (VREST) and AP threshold (VTHR). (C) Minimum current injection needed to evoke an AP (ITHR). The values of ITHR were normalized by ITHR in the absence of Retigabine (n = 4). (D) Example of a phase plot calculated from the APs shown on A. VREST was strongly affected by Retigabine (blue star), whereas VTHR remained fairly unaltered. Although the depolarization phase was affected by Retigabine (blue arrow), the late phase of repolarization (yellow arrow) remained unaltered. Arrows indicate the progress of the AP in time during the stimulation phase (purple arrow), depolarization phase (green arrow), and repolarization phase (orange arrow). The open blue arrow points at the times when APs reached their maximum voltage. The open yellow arrow points at the late repolarization phase. Error bars represent standard deviation. t test; n = 5. *, P < 0.002 with respect to control; **, P < 0.07 with respect to 1-µM Retigabine; ***, P < 0.0025 with respect to control; #, P < 0.072 with respect to control.
	Figure 6. Proposed kinetic scheme for the activity of KV7.2/KV7.3 and the effect of Retigabine. The scheme contains five global states, which are different with respect to each other depending on the activation status of the VSD and the pore domain (P). In the scheme, VSD represents the four voltage sensors of the channel. At negative potentials, the VSDs are in the deactivated state (VSDD) and the pore is, therefore, closed (PC0). Upon depolarization, the VSDs are activated (VSDA), allowing the pore to sojourn between a conductive and a nonconductive state (PO1 and PC1, respectively). Holding the membrane depolarized potentials keeps the VSDs in the VSDA state while allowing a second transition in the pore. As a result, the pore sojourns between a second pair of conductive and nonconductive states (PO2 and PC2, respectively) and the channel becomes Retigabine sensitive. Deactivation from these states is slower than from the pair PC1–PO1. Retigabine further decreases the rate of deactivation from the pair PC2–PO2 while leaving deactivation unaffected from the other pair.

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