Zhao R et al. (2020), Tethered peptide neurotoxins display two blocki...

XB-ART-57542

Sci Adv 2020 Mar 04;610:eaaz3439. doi: 10.1126/sciadv.aaz3439.

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Tethered peptide neurotoxins display two blocking mechanisms in the K+ channel pore as do their untethered analogs.

Zhao R, Dai H, Mendelman N, Chill JH, Goldstein SAN.

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We show here that membrane-tethered toxins facilitate the biophysical study of the roles of toxin residues in K+ channel blockade to reveal two blocking mechanisms in the K+ channel pore. The structure of the sea anemone type I (SAK1) toxin HmK is determined by NMR. T-HmK residues are scanned by point mutation to map the toxin surface, and seven residues are identified to be critical to occlusion of the KcsA channel pore. T-HmK-Lys22 is shown to interact with K+ ions traversing the KcsA pore from the cytoplasm conferring voltage dependence on the toxin off rate, a classic mechanism that we observe as well with HmK in solution and for Kv1.3 channels. In contrast, two related SAK1 toxins, Hui1 and ShK, block KcsA and Kv1.3, respectively, via an arginine rather than the canonical lysine, when tethered and as free peptides.

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	Fig. 1. T-HmK and HmK peptide in solution block KcsA-Shaker channels similarly.KcsA-Shaker was expressed in oocytes and studied by two-electrode voltage clamp (TEVC) to assess inhibition at equilibrium from a holding voltage of −80 mV with 300-ms test pulses to various voltages and a 5-s interpulse interval (n = 16 to 18 cells for each condition). Live oocytes were studied by ELISA and smTIRF microscopy as described in Materials and Methods. n = 12 oocytes for each condition. Values are means ± SEM. (A) Three SAK1 toxins: HmK, Hui1, and ShK peptide sequences aligned on their three disulfide bonds. (B) T-HmK was constructed as chimeric fusion protein with an N-terminal secretory signal sequence (SP; gray), the HmK sequence (green), a hydrophilic flexible linker with a c-Myc epitope tag (yellow), and a C-terminal GPI membrane anchor targeting sequence (orange). Same colors are used for marking T-HmK components in the above schematic. T-HmK is drawn free and binding to a potassium channel (blue) coexpressed in the same oocyte. The reaction volume that a T-HmK can visit (arrow) is determined by the lengths of the flexible peptide linker, GPI anchor, and the diameter of HmK toxin (Materials and Methods). (C) Representative raw current traces (steps from −80 to 60 mV) for KcsA-Shaker channels without (CTL) or with 0.25-ng T-HmK cRNA coinjection and after incubation with 2 μM anti-Myc to reverse the T-HmK blockade. (D) T-HmK linker variants (0.25 ng of cRNA) show different average KcsA-Shaker currents at 0 mV normalized to control, unblocked (CTL). T-HmK6 has 6 linker residues; T-HmK26 has 26 residues; T-HmK46 has 46 residues; and T-HmK66 has 66 residues. (E) Anti-Myc reverses blockade; KcsA-Shaker currents at 0 mV normalized to control, unblocked (CTL) with 0.25 or 0.5 ng of T-HmK46 cRNA. (F) Left: Single frame from a representative movie showing T-RFP (red fluorescent protein) on an oocyte membrane surface after 1-ng cRNA injection. Red circles mark single fluorescent spots. The white bar indicates 2 μm. Middle top: Schematic representation of T-RFP. Middle bottom: Montage of photobleaching time course of the single fluorescent particle indicated by the arrow in the left panel during continuous excitation to bleach the fluorophores. Every 12th frame is shown. Right bottom: Time course for photobleaching of RFP in the indicated particle, showing one stepwise change in fluorescence intensity (FI) (arrow). au, arbitrary units. Right top: The average number of single particles in 100-μm2 regions are 7 ± 1, 16 ± 2, or 43 ± 5 with 0.35, 0.5, or 1 ng of T-RFP cRNAs injected (n = 12 regions from three oocytes and four areas per oocytes for each condition). No fluorescent particles are observed in oocytes without T-RFP injection. (G) Increasing the amount of T-HmK cRNA injected increases surface expression on Xenopus oocytes. After 2 days of culture, surface expression of T-HmK was determined by ELISA using anti–Myc-HRP (horseradish peroxidase). The tethered control was without the toxin or c-Myc sequences (CTL). OD450, optical density at 450 nm. (H) The ELISA signals for T-RFP and T-HmK shows a stable relationship when the amount of injected cRNA increases. Two separate trials with six oocytes and five concentrations of T-RFP or T-HmK cRNA. (I) Concentration-response relationship for T-HmK (■) inhibition of KcsA-Shaker studied by TEVC and fit to the Hill relationship (Eq. 1). The determined Ki was 1.09 ± 0.01 nM with a Hill coefficient of 1.00 ± 0.01. n = 16 to 18 oocytes for each level of injected T-HmK cRNA. Coinjection of 0.35 ng of cRNA of T-HmK inhibited ~87% of the current, consistent with an effective T-HmK concentration of 7.4 ± 0.8 nM according to Eq. 1, similar to the ~8-nM surface concentration estimated by ELISA and smTIRF (table S1).
	Fig. 2. Measuring the kinetics of T-HmK blockade.KcsA-Shaker was expressed in oocytes and studied by TEVC as in Fig. 1 with a test potential of 0 mV with 16 to 18 cells for each condition. Values are means ± SEM. (A) The time course for block and unblock of KcsA-Shaker on acute application (bar) of 5 nM peptide HmK and washout. Every 10th data point is shown normalized to the unblocked current magnitude. (B) The time course for block and unblock of KcsA-Shaker on acute application (bar) of 100 mM TEA and washout. (C) The blocking kinetics of 5 nM HmK peptide was studied at various concentrations of TEA in bath solution. Each measured parameter Ki (●), reciprocal on rate association constant (▼), and off rate dissociation constant (▲) were normalized to the value without TEA. Each point represents the mean ± SEM from six oocytes. (D) Unblock of KcsA-Shaker on washout of 100 mM TEA allows subsequent block by 30 nM free HmK peptide. (E) Schematic representation of TEA competition and T-HmK block after TEA wash off. TEA blocks KcsA-Shaker (100 mM) from the outside like HmK and prevents T-HmK binding (left). Unblock by TEA is rapid (τ ~1.3 s), so full current is recorded on washout before significant association of T-HmK due to its slow on rate (τ ~ 33 s with 0.25 ng of T-HmK cRNA) (middle). Thereafter, T-HmK relaxes to equilibrium blockade (right). (F) With T-HmK, TEA wash-off KcsA-Shaker shows full current recovery and slow binding of T-HmK, similar to that seen with free peptide. The T-HmK association constant determined by a single exponential fit of the block time course was τon = 33 ± 4 s. (G) Effect of T-HmK concentration on blocking kinetics of KcsA-Shaker. Three T-HmK concentrations were studied after coinjection of 0.1, 0.25, and 0.5 ng of T-HmK cRNA estimated to represent an effective surface concentration of ~1, 3.5, and 17 nM (table S1). The apparent first-order rate constants for association (on rate, ▼) and dissociation (off rate, ▲) are plotted as a function of effective T-HmK concentration. Increasing the concentration of T-HmK from 1 to 17 nM increased the on rate of T-HmK from ~ 0.014 to 0.119 s−1, whereas the off rate was maintained at ~7.3 × 10−3 ± 1.1 × 10−3 s−1.
	Fig. 3. T-HmK screening mutagenesis and demonstration that Lys22 mediates voltage-dependent block of KcsA-Shaker channels.KcsA-Shaker was expressed in oocytes, and inhibition and surface expression of T-HmK and point variants were studied by TEVC or ELISA after coinjection of 0.5 ng of cRNAs. The equilibrium inhibition and kinetic blocking parameters of T-HmK after 0.25-ng cRNA coinjection and peptide HmK were studied by TEVC as in Fig. 2. n = 18 oocytes for each T-HmK concentration, and n = 6 for each level of HmK peptide. Values are means ± SEM. Some error bars are smaller than symbols. (A) Top: KcsA-Shaker currents at 0 mV measured as in Fig. 1E with T-HmK variants normalized to the unblocked condition (CTL). n = 16 to 18 cells for each variant. Bottom: ELISA performed as in Fig. 1G with T-HmK variants. Control was without the toxin or c-Myc sequences (CTL). n = 12 cells for each condition. (B) Effect of voltage on T-HmK blocking kinetics. The on rate is insensitive to voltage, whereas the off rate and, thus, the Ki are responsive, consistent with trans-ion knockoff. Each parameter was measured with test steps from −20 to 40 mV and normalized to its value at 0 mV, ▼, kon; ▲, koff; ■, Ki. Ki was determined from the fraction of unblocked current at equilibrium (Fun) according to Eq. 1. The association constant was determined by single exponential fits to the time course for T-HmK inhibition after TEA instant wash off. The dissociation rate constant was determined from Ki and kon according to Eq. 2. (C) Effect of voltage on blockade by T-HmK mutants. Neutralizing Lys22 (K22N) but not Arg24 (R24Q) removes the voltage dependence of toxin blockade. Ki for each variant was determined from −20 to 40 mV on the basis of the fraction of unblocked current at equilibrium (Fun) and plotted as a ratio to the value at 0 mV. (D) Raising intracellular KCl concentration speeds up the off rate of WT T-HmK but not when HmK-Lys22 is neutralized by mutation. Microinjection of 40 nl of KCl (0.25 to 1 M) increased the dissociation rate of T-HmK, whereas T-HmK–K22N was insensitive. Oocyte solute space has been estimated to be ~400 nl, and intracellular potassium concentration has been estimated to be ~90 mM; therefore, a 20-nmol KCl microinjection should raise intracellular potassium by ~50 to 140 mM, leading to an 11-mV shift in EK and a 50% increase in outward current, close to the observed values here of 10 ± 2 mV and a 45 ± 9% (n = 6 oocytes), as described before (11, 38). The new Ki were stable during the period of recording, that is, from 30 to 120 min after microinjection. (E) HmK peptides show the same Lys22 dependence of voltage blockade of KcsA-Shaker as T-HmK. Ki were determined from −20 to 40 mV on the basis of the fraction of unblocked current at equilibrium (Fun) and plotted as a ratio to the value at 0 mV. Association and dissociation time constants were determined by single exponential fits to the time course for block or unblock on acute application or washout of 5 nM HmK or 500 nM HmK-K22N.
	Fig. 4. Voltage-dependent block of hKv1.3 channels is mediated by Lys22 of HmK but by Arg24 of ShK.hKv1.3 was expressed in oocytes and studied by TEVC to assess equilibrium inhibition and kinetic blocking parameters of T-HmK after 0.5-ng cRNA coinjection, T-ShK after 0.1-ng cRNA coinjection, and peptide of ShK by the protocol described in Fig. 2. The Ki for T-HmK, T-ShK, peptide ShK, and their variants were determined from −20 to 40 mV on the basis of the fraction of unblocked current at equilibrium (Fun) and plotted as a ratio to the value at 0 mV. n = 18 oocytes for each condition of T-toxins, and n = 6 oocytes for each condition of peptide toxins. Values are means ± SEM. Some error bars are smaller than symbols. (A) Representative raw current traces for hKv1.3 (steps from −80 to 60 mV) without blocker (CTL), with 0.5-ng T-HmK cRNA coinjection, with 0.1-ng T-ShK cRNA coinjection, and after application of HmK or ShK peptides at 20 or 1 nM, respectively. (B) Neutralizing Lys22 (K22N) but not Arg24 (R24Q) removes the voltage dependence of T-HmK blockade of hKv1.3 channel. (C) Neutralizing Arg24 (R24Q) but not Lys22 (K22N) removes the voltage dependence of T-ShK blockade of hKv1.3 channel. (D) HmK peptides show the same Lys22 dependence of voltage blockade of hKv1.3 as T-HmK, parameters calculated as in Fig. 3E on acute application and washout of 20 nM HmK and 5 μM HmK-K22N. (E) ShK peptides show the same Arg24 dependence of voltage blockade of hKv1.3 as T-ShK, parameters calculated as in Fig. 3E on acute application and washout of 1 nM ShK, 5 nM ShK-K22N, and 1 nM ShK-R24Q.
	Fig. 5. HmK structure, interaction surface, and binding orientations.The HmK structure was determined from 328 distance and dihedral constraints derived from homo- and heteronuclear 2D NMR spectra (see Materials and Methods). (A) HmK structure (PDB accession code 6EI7). The 25 lowest-energy structures obtained from distance geometry/simulated annealing determination are shown. (B) Superposition of HmK (blue) with the crystal structure of ShK (4LFQ; gray) or the NMR structure of Hui1 (2N6B; orange) based on alignment of HmK/ShK residues 3 to 35 (Hui1, 2 to 34) shows that HmK retains an SAK1 scaffold. (C) Interaction surface of HmK mapped with T-toxin scanning as described in Fig. 3A. Orange indicates residues forming the primary toxin interaction surface (mutation caused ΔΔG > 2 kcal/mol). Yellow indicates Pro8 and Asn26 (mutation caused ΔΔG > 1.3 kcal/mol). The side chain of Lys22 is shown in dark orange. The two structures are separated by a rotation of 90o. (D) Left: Superposition of ShK and HmK highlighting ShK-Arg24 (gray)/HmK-Lys22 (blue) that mediate voltage dependence and ShK-Lys22 (gray)/HmK-Arg24 (blue) that do not. Right: Superposition of Hui1 and HmK highlighting Hui1-Arg23 (orange)/HmK-Lys22 (blue) that mediate voltage dependence and Hui1-Lys21 (orange)/HmK-Arg24 (blue) that do not. (E) Cartoon suggesting SAK1 toxin binding orientations in the hKv1.3 external vestibule with Lys or Arg near the conduction pore. Left: Pose 1 for HmK with Lys22 toward K+ in the pore. Middle: Pose 2 for ShK with Arg24 near K+ in the pore. Right: Pose 1 for ShK with Arg24 flexing to interact with K+ in the conduction pore.

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