Horne AJ , Peters CJ , Claydon TW , Fedida D .

Canadian Institutes of Health Research

	Figure 1. An alignment of the S3–S4 linkers of Shaker and Kv1.2 highlights similarities in the region. Gray shading denotes the S3 and S4 regions of the protein. Residues assayed for voltage-dependent fluorescence (Fig. 3) are underlined, and the residue denoted by the closed circle corresponds to Kv1.2 A291C (Shaker A359C).
	Figure 2. Voltage-dependent Shaker and Kv1.2 conductance and fluorescence deflections. Typical current traces (A and D) and fluorescence signals (B and E) are shown for Shaker C245V A359C and Kv1.2 C181 A291C for 100-ms pulses between −120 and 60 mV from −80 mV. The dashed line in E marks the approximate division of the two observed components of A291C fluorescence for depolarizations positive to 0 mV. (C and F) Normalized conductance-voltage (G-V; closed symbols) and total fluorescence-voltage (F-V; open symbols) relationships were calculated from data obtained at the end of each 100-ms pulse (mean ± SEM; n = 7–8), and data were well fit with Boltzmann equations. The V1/2 values for Shaker C245V A359C were −12.9 ± 1.6 mV and −39.5 ± 1.5 mV for G-V and F-V, and slope factors (k) were 17.8 ± 1.1 mV and 22.3 ± 1.4 mV, respectively. The Kv1.2 A291C G-V had a V1/2 and slope factor of −11.2 ± 1.6 mV and 22.4 ± 0.9 mV, respectively, whereas the F-V relationship had a V1/2 and k of −62.9 ± 1.2 mV and 23.2 ± 1.0 mV.
	Figure 3. A scan of the Shaker and Kv1.2 S3–S4 linkers reveals differences in fluorescence phenotypes. (A and B) Representative fluorescence traces collected in a cysteine scan of five consecutive homologous residues in the S3–S4 linker and N-terminal end of S4 in Shaker (A) and Kv1.2 (B). Cells expressing these constructs were held at −80 mV and depolarized to 60 mV for 100 ms. (C–F) Normalized G-V (closed symbols) and F-V (open symbols) relationships for four of the five Shaker (squares) and Kv1.2 (circles) constructs expressing changes in fluorescence upon depolarization, as labeled in the top left corner of each panel. For Kv1.2 S289C (D), the gray circles and accompanying fit denote the Boltzmann fit to the voltage-dependent fluorescence observed between −140 and −20 mV. Mean half-activation and slope data obtained from the fits to all four mutant constructs for each channel can be found in Table I. Data are shown as mean ± SEM and are the mean of three to eight cells collected from each mutant.
	Figure 4. Holding potential affects the directionality of Kv1.2 fluorescence deflections but does not affect the overall F-V relationship. (A and B) Representative deflections of Kv1.2 C181 A291C held at −120 (A) and −50 mV (B) in response to 100-ms changes in voltage between −150 and 60 mV. (C) Normalized G-V (closed symbols) and total F-V (open symbols) relationships for Kv1.2 C181 A291C from three different holding potentials (mean ± SEM; n = 3–5), with F-V relationships adjusted to account for upward deflections. From −120 mV (triangles), the G-V had a V1/2 of −10.7 ± 2.5 mV and a slope factor of 24.8 ± 0.9 mV and corresponding values of −69.5 ± 2.0 mV and 22.7 ± 1.1 mV, respectively, for the F-V. V1/2 and slope factor data from −80 mV (circles) were −2.2 ± 2.3 mV and 24.6 ± 0.7 mV for the G-V and −69.1 ± 4.5 mV and 20.8 ± 0.8 mV for the F-V. At −50 mV (squares), the V1/2 and slope factor for the G-V and F-V were 2.6 ± 0.8 mV and 24.2 ± 1.1 mV and −69.9 ± 5.8 mV and 22.6 ± 1.5 mV, respectively. (D) Shaker A359C G-V and F-V relationships from the same three holding potentials as in C (mean ± SEM; n = 7–8). From −120 mV (triangles), the G-V had a V1/2 of −22.2 ± 2.1 mV and a slope factor of 15.8 ± 1.6 mV and corresponding values of −35.0 ± 4.5 mV and 27.5 ± 1.6 mV, respectively, for the F-V. V1/2 and slope factor data from −80 mV (circles) were −22.1 ± 1.9 mV and 15.9 ± 1.7 mV for the G-V and −39.8 ± 4.0 mV and 29.1 ± 2.1 mV for the F-V. At −50 mV (squares), the V1/2 and slope factor for the G-V and F-V were −12.7 ± 1.9 mV and 15.6 ± 1.7 mV and −79.6 ± 4.8 mV and 32.4 ± 1.5 mV, respectively.
	Figure 5. Kv1.2 A291C voltage-dependent fluorescence is well characterized by a double exponential function. (A) Fit of the Kv1.2 A291C fluorescence signal at 60 mV to a double exponential shows that ∼40% of the signal amplitude results from a fast movement, with a time constant of 1.3 ms. The slow phase, comprising 60% of the total signal, is slower by an order of magnitude, 23.9 ms in this example. (B) Mean ± SEM time constants of the fast and slow fluorescence signal components (n = 14–20) compared with time constants of ionic current activation fit from ∼50% of maximal activation. (C) Normalized F-V relationships of the fast and slow components of Kv1.2 A291C fluorescence normalized and plotted alongside the G-V relationship (n = 11). The normalized fast phase, fit to a Boltzmann distribution, had a V1/2 and slope factor of −39.5 ± 2.0 mV and 15.6 ± 1.0 mV. The voltage dependence of the slow phase was best fit with a double Boltzmann function, with the first component having a V1/2 and k of −73.9 ± 1.4 mV and 12.0 ± 0.5 mV (amplitude = 88.7 ± 2.3%) followed by a second component (11.2 ± 2.5%) with respective V1/2 and slope factors of 44.3 ± 4.2 mV and 11.6 ± 3.0 mV. (D) Holding potential–dependent separation of the fast and slow fluorescence components. Representative fluorescence traces are shown for Kv1.2 C181 A291C channels depolarized to −30 mV (gray traces) or 60 mV (black traces) from holding potentials (HP) of either −80 or −50 mV as labeled. The two vertical lines to the left of the fluorescence records show the contributions of fast and slow quenching components for depolarizations to 60 mV.
	Figure 6. Correlation of the fast component of Kv1.2 fluorescence quenching with gating charge movement. (A) Representative gating currents for Kv1.2 WT, recorded from transiently transfected tsA201 cells. Data were recorded from 12-ms pulses from −80 to 60 mV at 10-mV increments; only every third voltage is shown here for clarity. (B) Overlay of the mean normalized charge-voltage (Q-V) relationship with the fast F-V relationship from Fig. 5 C. The Q-V relationship V1/2 and k values were −31.5 ± 2.0 mV and 11.5 ± 0.6 mV, respectively. (C) Superposition of fluorescence (gray) and cumulative gating charge (black) versus time in Kv1.2 channels. Data are shown for a range of depolarizations from −50 to 60 mV, as labeled, for the initial 8 ms of depolarization in the case of the fluorescence data and are normalized to the respective maximum values. The fluorescence quenching at this potential is inverted to more closely compare with the gating charge data. (D) Mean time constants of the integrated gating charge movement compared with the fast fluorescence quenching of A291C. Gating charge data were fit to a double exponential function, and the mean ± SEM data (n = 10–15) of the fast (closed squares) and slow (open squares) components were plotted as a function of voltage. Time constants for the fast quenching event (open circles) are as plotted in Fig. 5 B.
	Figure 7. Slow fluorescence return upon hyperpolarization correlates with deactivation of ionic current. (A) Kv1.2 A291C currents (top) and fluorescence (bottom) traces at −120, −60, 0, and 60 mV from a holding potential of −120 mV. The right panels show an enlarged view of the tail currents and off-fluorescence emissions. (B) Overlay of ionic tail current (black lines) and slow off-fluorescence quenching (gray) for Kv1.2 A291C at the three potentials labeled after a depolarization to 20 mV. Scale bars are as shown, left to right, for the corresponding holding potentials. (C) Deactivation and slow off-fluorescence time constants for Kv1.2 A291C at the three holding potentials shown in B as a function of prepulse potential (n = 3–5). Mean data are shown every 20 mV for clarity. (D) Representative current and fluorescence records from a dual pulse (P1–P2) protocol with varying interpulse recovery time. Data are shown for 100-ms pulses from −80 to 60 mV with interpulse intervals of 6.25, 25, 100, and 250 ms. For clarity, data are only shown up to the end of the second depolarizing pulse of intermediate records. The gray arrow shows the instantaneous level of ionic current at P1; the gray dashed line is an inverted fit of the slow component of off-fluorescence from P1. (E) Overlay of the slow off-fluorescence component in D with the normalized initial P2 current amplitudes (black diamonds). The black line is a single exponential fit to the ionic current data. Mean time constants for the fits to individual datasets and the off-fluorescence component are shown in the panel (n = 5).
	Figure 8. Kv1.2–Kv1.5 chimera channels lack the slow fluorescence quenching at negative potentials. (A) Cartoon representation of Kv1.2 A291C (top), Kv1.5 WT (bottom), and the two chimeric channels (middle cartoons). Open squares (and their connecting segments) originate from Kv1.2, and slashed squares are Kv1.5 segments. A291C is located approximately with closed circles at the N-terminal S4. (B) Representative fluorescence traces from the Kv1.5-S12L-Kv1.2 A291C chimera. Data were collected using the protocol outlined in Fig. 2. (C) Overlay of representative fluorescence deflections for Kv1.2 A291C and Kv1.5-S12L-Kv1.2 (S12L) A291C for depolarizations to 30 mV normalized to the fast fluorescence quenching components. The inset shows overlays of deactivating ionic tail currents and off-fluorescence of Kv1.5-S1S2L-Kv1.2 A291C (top) and Kv1.2 A291C (bottom), with scale bars as noted. (D) Mean normalized G-V (closed symbols) and F-V (open symbols) relationships for the Kv1.5/Kv1.2 chimera channels compared with Kv1.2 A291C, shown ±SEM (n = 10–13). Boltzmann fits to the data from Kv1.5-S12L-Kv1.2 A291C (S12L) gave V1/2 and k values of 2.6 ± 2.6 mV and 23.9 ± 0.9 mV and −40.3 ± 3.1 mV and 28.4 ± 2.6 mV for G-V and F-V, respectively; the fits to Kv1.5-S23L-Kv1.2 A291C (S23L) were 7.3 ± 1.9 mV and 23.9 ± 0.7 mV for the G-V relationships and −40.9 ± 2.2 mV and 23.4 ± 2.3 mV for the F-V relationships. Kv1.2 A291C values are as reported in Fig. 4. (E) Voltage-dependence plots of the fast fluorescence component of Kv1.5-S23L-Kv1.2 A291C with that of Kv1.2 A291C from Fig. 5 C. V1/2 and k values were −40.3 ± 2.2 mV and 19.5 ± 1.7 mV for the chimera channel (n = 15–17). (F) Mean normalized F-V relationship of the fast component of Kv1.5-S23L-Kv1.2 A291C emission, overlaid with the Kv1.2 Q-V relationship (closed circles) from Fig. 6 B.
	Figure 9. Effect of Shaker S1–S2 linker replacement on Kv1.2 fluorescence. (A) Representative fluorescence records from a Kv1.2-Shaker S1–S2 linker A291C chimera channel (Kv1.2Sh12L). Traces are shown for depolarizations to potentials as labeled, using the same protocol as outlined in Fig. 2. (B) Mean F-V relationship for Kv1.2Sh12L (triangles) compared with Kv1.2 A291C (circles) and Shaker A359C (squares) as previously shown in Fig. 2. For Kv1.2Sh12L, V1/2 and k values for the Boltzmann fit were −56.0 ± 2.5 mV and 15.5 ± 0.8 mV (n = 7).
	Figure 10. TMRM attached to residues in the S1–S2 linker of Kv1.2 report only slow changes in fluorescence emission in response to voltage. (A) An alignment of best fit for the amino acid residues of the S1–S2 linker region (underlined) of Shaker, Kv1.2 and Kv1.5. Residues in gray correspond to portions of the S1 and S2 helices. Residues tested for fluorescence are boxed, and those giving voltage-dependent deflections are in bold. (B and C) Representative fluorescence emissions recorded at 60 mV from I187C (B) and T219C (C) channels. (D) Mean G-V (closed symbols) and F-V (open symbols) relationships for I187C (diamonds) and T219C (triangles; n = 6–15) compared with Kv1.2 A291C fast and slow fluorescence components from Fig. 5 C (open circles and squares, respectively). Boltzmann fits to I187C data gave V1/2 and k values of −50.6 ± 1.4 mV and 10.6 ± 0.5 mV for F-V and −17.7 ± 1.9 mV and 27.3 ± 1.0 mV for G-V. T219C half-activation potential and slope factor values were −15.5 ± 2.6 mV and 22.8 ± 0.3 mV and −70.8 ± 5.9 mV and 18.3 ± 2.0 mV for the G-V and F-V relationships. Error bars represent mean ± SEM.
	Figure 11. Proposed model of Kv1.2 activation based on TMRM fluorescence. (A) Closed state structure of Kv1.2 A291C-TMRM based on the closed state model of Pathak et al. (2007), shown from the top. Only one voltage-sensing domain (yellow) and the pore domain from an adjacent subunit (green) are shown. Residues tested are labeled and highlighted based on their ability (blue) or inability (orange) to yield voltage-dependent fluorescence; the A291C mutation is shown in red. The TMRM molecule is modeled within the external aqueous vestibule between S4 and S1–S3 as a spherical structure of carbon (green), oxygen (red), and nitrogen (blue) atoms. (B) Open state structure of Kv1.2 A291C-TMRM. The view shown, looking down on the channel, is similar to that in A for the closed state channel relative to the pore domain, in particular S5. (C and D) Side views of the closed and open state structures of Kv1.2 A291C-TMRM seen in A and B. Transmembrane helices of one subunit are as labeled in each panel.

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