Zhao R , Qasim A , Sophanpanichkul P , Dai H , Nayak M , Sher I , Chill J , Goldstein SAN .

R01 HL159711 NHLBI NIH HHS , U54 GM087519 NIGMS NIH HHS , R01 GM111716 NIGMS NIH HHS , R61 AT012544 NCCIH NIH HHS

                focal epilepsy
                multiple sclerosis
                episodic ataxia

	FIGURE 1 SAK1 toxins display two blocking mechanisms in the K+ channel pore. (A) Sequence alignment of AETX-K, HmK, and Hui1. Pore occluding residues were labeled blue. Identical residues between AETX-K and HmK were marked with gray color. (B) Cartoon suggesting SAK1 toxin binding orientations in the Kv1.1 channel or KcsA channel external pore vestibule with Lys or Arg near the conduction pore. Left: AETX-K with Lys23 toward K+ in the Kv1.1 channel pore. The epilepsy-associated gain-of-function mutation L296F (shown in red) locates at the S4 transmembrane segment in the voltage sensor domain of the Kv1.1 channel. Middle: HmK with Lys22 toward K+ in the KcsA channel pore as described before. Right: Hui1 with Arg23 toward K+ in the KcsA channel pore as described before.
	FIGURE 2 AETX-K peptide blocks Kv1.1 with low picomolar affinity. Kv1.1 channels were expressed in oocytes and studied by two-electrode voltage clamp (TEVC) to assess equilibrium inhibition and kinetic blocking parameters using a holding voltage of −100 mV, 300-ms test pulses, and a 5-s interpulse interval, n = 5–12 cells for each condition. Values are mean ± SEM. Some error bars are smaller than symbols. (A) Representative current traces for Kv1.1 channels at steady state before (Control), in the presence of 50 pM AETX-K, and after toxin washout (Wash) with steps of 20 mV from −100 to 80 mV. (B) The time course for block and unblock of Kv1.1 on acute application (bar) and washout of 50 pM AETX-K. Peak currents recorded at 0 mV; every sixth point is shown. (C) Dose–response relationships for AETX-K inhibition of Kv1.1 studied as in B and fit to the Hill equation (Materials and Methods).
	FIGURE 3 Structure of AETX-K as determined by NMR. (A) Superposition of 23 low-energy NMR structures of AETX-K. Shown are AETX-K with the first helix head-on (left) and in channel-binding pose with Lys23 pointing downward (right). Disulfide bonds are highlighted in yellow and sidechains of Lys21 and Lys23 in blue. (B) Electrostatic surface rendering of toxins Hui1 (left), AETX-K (middle) and HmK (right) in aligned orientations according to interpolated charge (red/blue = negative/positive). Sidechains of Lys21 and Lys23 are highlighted as blue spheres.
	FIGURE 4 Interaction of AETX-K with KcsA and Kv1.1 are revealed by NMR. (A) Interaction of KcsA (left) and chimeric Kv1.1 (right) with AETX-K followed by 15N,1H-HSQC spectra without the LPN-embedded channel (black) and with 0.33 eq. (red) and 1.0 eq. (green) of channel. Increased weakening of peak intensity reflects stronger affinities. (B) Summary of intensity decreases along the AETX-K sequence. Gray (dark gray) bars indicate the normalized intensity (in comparison to peaks prior to addition of channel) at 0.33 (1.00) mol:mol channel equivalents. Errors were estimated from S/N levels in all spectra.
	FIGURE 5 AETX-K-Lys23 is responsible for voltage-dependent dissociation. Kv1.1 channels were expressed in oocytes and studied by TEVC as indicated in Figure 2. n = 5–12 cells for each condition. Values are mean ± SEM. Some error bars are smaller than symbols. (A) Effect of voltage on AETX-K blocking kinetics of Kv1.1 channels. Each parameter was measured with test steps from −20 to 40 mV and normalized to its value at 0 mV. Association and dissociation rate time constants were determined by single-exponential fits to the time course for block or unblock on acute application or washout of 50 pM AETX-K. The inhibition constant Ki was calculated from the fraction of unblocked current at equilibrium and the rate constants (Materials and Methods). The change in Ki with voltage is due to the altered dissociation rate. (B) Effect of voltage on dissociation rate of Kv1.1 by AETX-K mutants. Koff for each toxin was determined from −20 to 40 mV based on the single-exponential fits to the time course for unblock on acute application or washout of 50 pM AETX-K, 5 μM AETX-K-K21N and 500 nM AETX-K-K23N and plotted as a ratio to the values at 0 mV
	FIGURE 6 AETX-K inhibits Kv1.1-L296F channels effectively. Kv1.1 and Kv1.1-L296F channels were expressed in oocytes and studied by TEVC as indicated in Figure 2. n = 5–12 cells for each condition. Values are mean ± SEM. Some error bars are smaller than symbols. (A) Representative current traces for Kv1.1 and Kv1.1-L296F channels with steps of 10 mV from −100 to 80 mV. (B) Conductance-voltage (G-V) relationship for Kv1.1 and Kv1.1-L296F. Kv1.1-L296F channels showed a −59 ± 4 mV shift in half-maximal activation voltage (V1/2) compared to wild-type Kv1.1. Curves fit to a Boltzmann equation (Materials and Methods). (C) Dose–response relationships for AETX-K inhibition of Kv1.1-L296F studied as in Figure 2 and fit to the Hill equation (Materials and Methods). (D) Voltage–voltage (I-V) relationship for Kv1.1, Kv1.1-L296F, and Kv1.1-L296F with incubation of 4 pM AETX-K.
	FIGURE 7 AETX-K blocks mixed populations of Kv1.1-Kv1.2 channels. Kv1.1 and Kv1.2 channels, and Kv1.1-Kv1.2 heterotetrameric channels formed by a cRNA ratio of 50%:50% were expressed in oocytes and studied by TEVC as indicated in Figure 2. n = 3–12 cells for each condition. Values are mean ± SEM. Some error bars are smaller than symbols. (A) Representative current traces for Kv1.1, Kv1.1-Kv1.2, and Kv1.2 channels with steps of 10 mV from −100 to 80 mV. (B) G-V relationship for Kv1.1, Kv1.1-Kv1.2, and Kv1.2. Curves fit to a Boltzmann equation (Materials and Methods). (C) After co-expression, Kv1.1 and Kv1.2 subunits assemble into tetrameric assembled channels with five different subunit stoichiometries. The theoretical percentages of expression of the five different stoichiometries of assembled channels can be calculated with the binomial equation (Materials and Methods), assuming that equal numbers of Kv1.1 and Kv1.2 subunits randomly assemble into tetrameric channels, each with equal probability to reach the membrane surface. (D) Representative current traces for Kv1.1, Kv1.1-Kv1.2, and Kv1.2 channels at steady state in the presence of 0.1 nM AETX-K with steps of 10 mV from −100 to 80 mV. (E) Kv1.1, Kv1.1-Kv1.2, and Kv1.2 currents at the end of a test pulse to 0 mV with 100 pM AETX-K. (F) Apparent block of Kv1.1-Kv1.2 currents with 0.1, 10, 100 and 1000 nM AETX-K. The Ki for channels with different subunit stoichiometries are summarized in Table 3.

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