Wang G , Shahidullah M , Rocha CA , Strang C , Pfaffinger PJ , Covarrubias M .

AA07463 NIAAA NIH HHS , P01 NS37444 NINDS NIH HHS , R01 NS032337-06 NINDS NIH HHS , R01 NS032337-07 NINDS NIH HHS , R01 NS032337-08 NINDS NIH HHS , R01 NS032337-09A1 NINDS NIH HHS , R01 NS032337-10 NINDS NIH HHS , R01 NS032337-11 NINDS NIH HHS , R01 NS032337-12 NINDS NIH HHS , R01 NS31583 NINDS NIH HHS , R01 NS32337 NINDS NIH HHS , R01 NS032337 NINDS NIH HHS , P01 NS037444 NINDS NIH HHS , R01 NS031583 NINDS NIH HHS , T32 AA007463 NIAAA NIH HHS

	Figure 1. Time-dependent inhibition of Kv4 channels by thiol-specific reagents applied to the intracellular side of the membrane. (A–H) Currents were evoked by a step depolarization from −100 mV to +50 mV at 3-s intervals. The intracellular side of inside-out macropatches from Xenopus oocytes was exposed to the thiol-specific reagents (200–600 μM MTSET, 200 μM MTSES, 2 mM NEM). The current traces are averages of ∼7–20 individual responses recorded before the reagent application (black) and after the inhibition approached steady-state (red). Reagent exposure in the corresponding inhibition time courses (B, D, F, and H) is indicated by a black horizontal bar. Each symbol type represents a separate macropatch (B, n = 2; D, n = 3; F, n = 2; H, n = 3; J, n = 4). Red lines in these graphs are the best fits assuming an exponential decay. The mean values of the derived second-order rate constants (1/(τ × [reagent])) were 248, 58, 37, and 8 M−1s−1, for B, D, F, and H, respectively. (I and J) When MTSET (600 μM) was present in the pipette (external solution), the current remained stable. The black and red traces correspond to the average currents obtained during the first and second half of the experiment, respectively (black and red bars).
	Figure 2. Mutagenesis of intracellular cysteines in Kv4.1 channels. (A) Schematic topology of the Kv4.1 pore-forming subunit. Filled red circles mark the approximate positions of the 14 cysteine residues likely exposed to the intracellular milieu. (B) Nomenclature of the Kv4.1 mutant channels. Alanine was substituted for cysteine at the indicated positions. CZn indicates that the marked cysteine contributes to Zn2+ binding in the crystal structure of the isolated T1 tetramer.
	Figure 3. Functional properties of Cys-depleted Kv4.1 channels. The Kv4.1 wild type and the mutant C11xA (Fig. 2) were functionally expressed in the absence of auxiliary subunits. (A and B) In the cell-attached configuration, macropatch outward currents were evoked by step depolarizations from −100 mV to command voltages ranging between −80 and +60 mV in 20-mV increments. (C) Voltage dependence of the peak conductance. Filled and hollow symbols correspond to wild-type and C11xA channels, respectively. The solid lines are the corresponding best-fit fourth-order Boltzmann functions with the following best-fit parameters for wild type: Va = −53.6 ± 1.8 mV, V1/2 = −10.9 ± 2.9, k = 25.6 ± 1.2 mV (n = 5); and for C11xA: Va = −63.9 ± 4.5 mV, V1/2 = −8.1 ± 6.4 mV, k = 33.6 ± 1.3 mV (n = 5). (D) The time constants of inactivation were determined from the best biexponential fits describing the decay of the currents at the examined membrane potentials (Beck and Covarrubias, 2001; Shahidullah et al., 2003). (E) The ratio of the amplitudes (AFAST/ASLOW) of the biexponential fits plotted against the examined membrane potentials. Note that the kinetic parameters of current decay are only modestly affected by the C11xA mutation.
	Figure 4. Functional properties of Cys-depleted Kv4.1 channels coexpressed with auxiliary subunits. All mutants examined here were expressed as ternary complex including the Kv4.1 pore-forming subunit, DPPx-s and KChIP-1. (A–C) In the cell-attached configuration, currents were evoked by step depolarizations from −100 mV to command voltages ranging between −90 and +110 mV in 20-mV increments. (D) Kv4.1-C11xA, Kv4.1-C12xA, Kv4.1-C13xA, and Kv4.1-C14xA exhibit modestly accelerated inactivation but maintain preferential closed-state inactivation as evident from the voltage dependence of the time constant of inactivation (Jerng et al., 2004b). The solid lines are the best-fit exponential growth functions (Table I). (E) Kv4.1-C11xA, Kv4.1-C12xA, Kv4.1-C13xA, and Kv4.1-C14xA do not exhibit significantly altered voltage dependence of the peak conductance. The solid lines are the corresponding best-fit fourth-order Boltzmann functions (Table I).
	Figure 5. Effects of internal Zn2+ or TPEN on Kv4 channels with intact (C11xA) or disrupted (C14xA) T1 Zn2+ sites. Mutant channels were expressed as described in Fig. 4 legend. (A and B) Outward Kv4.1-C11xA currents evoked by a step depolarization to +80 mV from a holding potential of −100 mV. These currents were recorded from inside-out patches before (black) and after (red) the intracellular application of Zn2+ or TPEN at the indicated concentrations. (C) Bar graph comparing the normalized peak current (at +80 mV) before and after the application of Zn2+ or TPEN. (D) Bar graph comparing the effects of Zn2+ and TPEN on the time constant of inactivation at +80 mV (Fig. 4). The Zn2+ and TPEN experiments were conducted separately; therefore, the data are presented in two groups. Note that independently of the integrity of the T1 Zn2+ site, the peak current and time constant of inactivation decreased upon TPEN application (see text).
	Figure 6. Inhibition of Kv4.1-wild type and Zn2+ site mutants by internal MTSET. Mutant channels were expressed as described in Fig. 4 legend. (A, C, E, and G) Time-dependent inhibition of wild type, C11xA, C12xA, C13xA, and C14xA (Fig. 2 for mutant nomenclature) by internally applied MTSET (arrow, 200 μM). The y axis is the normalized peak current (peak current after MTSET/peak current before MTSET). These experiments were conducted as explained in Fig. 1 legend. The solid black line through the data points corresponds to the best-fit exponential. The second-order rate constants are indicated within the corresponding panels. (I) When all internal cysteines were mutated to alanines (C14xA), there was no inhibition by MTSET. Red symbols and gray bars represent the mean ± SEM from the number of independent measurements indicated in the corresponding panels. (B, D, F, H, and J) Representative current traces (corresponding to the left panels in each row) evoked by a step depolarization from −100 to +80 mV (inside-out patch configuration). The traces are averages (∼7–20 traces) taken before (black) and after (red) approaching the steady-state level of the inhibition.
	Figure 7. Inhibition of Kv4.1 Zn2+ site mutants by intracellular MTSET in the presence of Zn2+ or a Zn2+-specific chelator. The experiments were conducted as explained in Fig. 6 legend. (A) MTSET-induced inhibition in the presence of 10 μM ZnCl2 in the internal solution (no EGTA added). The solid line across the symbols corresponds to the best-fit exponential function (1/τ = 0.067 mM−1s−1; A∞ = 0.2; A∞ is the estimated level of the remaining current). (B) MTSET-induced inhibition in the presence of 20 μM TPEN in the internal solution. The solid line across the symbols corresponds to the best-fit exponential function (1/τ = 0.050 mM−1s−1; A∞ = 0.16). The insets in A and B display the currents before (thick line) and after (thin line) application of MTSET to the internal side of the inside-out patch. The dashed line marks the zero current level. (C) Bar graph summarizing the MTSET second-order inhibition rate constants (1/(τ × [MTSET])) for various Kv4.1 mutants (Fig. 2) examined under control conditions or in the presence of ZnCl2 or TPEN.
	Figure 8. Biochemical evidence of the chemical modification of Kv4.2 and Kv4.2-T1 by MTSEA-biotin. (A) Membrane fragments containing Kv4.2 and KChIP3 (Kv4.2:KChIP3, 1:3) were reacted with MTSEA-biotin for 20 min at room temperature, and then electrophoresed and blotted with either anti-Kv4.2 or streptavidin-HRP (MATERIALS AND METHODS). (B) Likewise, the purified T1 domain of Kv4.2 was also reacted with the biotinylated MTSEA reagent and screened with streptavidin-HRP. The indicated molecular weights correspond to those of Kv4.2 a-monomer (67 kD) and the monomeric Kv4.2-T1 protein (14 kD). (C) FPLC profile of the Kv4.2-T1 protein before (black) and after (red) treatment with MTSEA-biotin (0.5 mM). AU280, normalized absorbance units at 280 nM. The expected elution times of the T1 tetramer and the T1 monomer are schematically marked above the abscissa.
	Figure 9. State-dependent inhibition of Kv4.1 wild type and Kv4.1-C11xA currents by internally applied MTSET. The Kv4.1 pore-forming subunits were expressed alone and the outward currents were recorded in the inside-out patch configuration. The acquisition program controls the coordinated application of voltage steps and MTSET concentration jumps (MATERIALS AND METHODS). (A) MTSET (200 μM) was applied when the membrane was held at −100 mV (resting channels), and the test current was evoked by the indicated pulse protocol. (B) MTSET (200 μM) was applied during the step depolarization to +80 mV as illustrated in the figure (activated channels). For both resting and activated channels, the duration of the reagent concentration jump was 7 ms. (C) Time courses of the inhibition by MTSET of resting (hollow symbols) and activated (filled symbols) channels. The solid lines are the best-fit exponential decays with the following best-fit parameters: 1/τ = 20 mM−1s−1 and A∞ = 0.4, for activated channels; 1/τ = 0.9 mM−1s−1 and A∞ = 0.2, for resting channels. The observed resting state rate constant is not an accurate estimation of the overall modification rate constant because the brief time frame of the experiment (0–0.2 mM × s) severely limits the observed fraction of the slow decay (see below). However, the current data cannot rule out the possibility of a small fast component (≤10%) in the modification time course of the resting channels. (D) Bar graph summarizing the second-order rate constants of the modification by MTSET (n = 4–6). For a more accurate estimation of the slow rate constant from resting channels, the duration of the MTSET concentration jump was 240 ms and the MTSET concentration was 400 μM. Under these conditions, ≥90% of the decay was exponential.
	Figure 10. A structural model of the Kv4-T1 domain and the contribution of T1–T1 intersubunit interface to the gating state–dependent inhibition of the Kv4 channel by MTSET. (A) Model of the Kv4 tetramer depicting a theoretical model of the transmembrane α-core (Durell et al., 2004) and the likely location of the intracellular T1 domain (red). The depicted 3-D model of the T1 domain is based on the crystal structure of the isolated Kv4-T1 domain (Nanao et al., 2003). (B) Magnified views of the T1 domain model. In both, the lateral (left) and top (right) views, the blue spheres represent the location of Zn2+ atoms in the T1–T1 intersubunit interface as found in the crystal structure. The four layers of the T1 scaffolding are indicated next to the lateral view as L1–L4. Note that the Zn2+ site is located between L3 and L4. In the intact channel, L4 is directly connected to the NH2-terminal end of the transmembrane S1 segment, which links T1 with the voltage-sensing regions of the channel. (C) Magnified view of a single interfacial Zn2+ site in T1. The coordinating amino acid side chains are explicitly shown in a scaled ball and stick representation, and the outlined circle (dashed line) represents the Zn2+ atom. H104, C131, and C132 are from the same subunit, and C110 is from the neighboring subunit. H104 is behind the backbone of C131 and C132. A standard color scheme is used to represent the relevant atoms (sulfur atoms in yellow). Note that the peptide bond between C131 and C132 is also shown. (D) Working hypothesis explaining the gating state dependence of the inhibition of Kv4 channels by MTSET. In the resting state (at hyperpolarized voltages), at least one thiolate group (e.g., C131) exhibits low accessibility to MTSET. Upon activation by a strong depolarization, a concerted clockwise displacement of L4 exposes the Zn2+ site thiolate groups to MTSET. The data suggest that modification of a single thiolate group in the T1–T1 interface would be sufficient to explain the inhibition of the channel (see text). A possible location of Zn2+ in these models is indicated by the dashed perimeter of a circle in the T1–T1 interface. The exact alternative architecture of the T1 Zn2+ site that leaves at least one free thiolate in the T1–T1 interface is not known (see text).

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