Li-Smerin Y , Swartz KJ .

ZIA NS002945-13 NINDS NIH HHS , ZIA NS002945-13 Intramural NIH HHS

	Figure 2. Inhibition of the drk1 K+ channel by Hanatoxin. (A) Families of current records obtained from two-electrode voltage-clamp recording of oocytes expressing the wild-type (left) and two mutant drk1 K+ channels (F274R, middle; E277K, right), both in the absence and presence of 1 μM toxin. Currents were elicited by voltage steps beginning at −50 mV and incrementing in 5-mV steps from a holding voltage of −80 mV. 15 traces are shown for wild-type and F274R families (−50 to +20 mV), while 17 records are shown for the E277K family (−50 to +30 mV). Tail currents carried by Rb+ were elicited by repolarization to −50 mV. All traces are shown without subtraction of leak or capacitive currents. (B) Tail current voltage-activation relations for wild-type (left), F274R (middle), and E277K (right). Tail current amplitudes were measured 2–3 ms after repolarization in the absence (○) or presence (•) of 1 μM Hanatoxin. Same oocytes as in A. (C) Fraction of uninhibited tail current plotted against test voltage at different toxin concentrations for wild-type, F274R, and E277K. I is the tail current measured in the presence of toxin and I0 is the tail current measured in the absence of toxin. Data are shown as mean ± SEM for three to six cells at each toxin concentration.
	Figure 1. Topology and architecture of voltage-gated K+ channels. (A) Sequence of the drk1 K+ channel for the region starting at the NH2 terminus of S3 and ending at the COOH-terminal of S4 and the putative topology of a single α subunit. Yellow highlighting indicates the three positions (I273, F274, and E277) where point mutations have been found to alter the binding affinity of Hanatoxin (Swartz and MacKinnon 1997b). The putative demarcations of the S3 and S4 transmembrane segments, indicated by underlining, are based on Kyte-Doolittle hydrophobicity analysis. The cylinders above the sequence indicated the approximate positions of three α helices. The solid cylinders (for S3 and S4) are based on α-helical periodicity detected using alanine- and tryptophan-scanning mutagenesis (Hong and Miller 2000; Li-Smerin et al. 2000a). The dotted cylinder positioned near the COOH terminus of S3 and the S3–S4 linker was proposed based on helical periodicity detected with alanine-scanning mutagenesis (Li-Smerin et al. 2000a). The topology of a single K+ channel subunit shown (bottom) illustrates the first four transmembrane segments (S1–S4, in black) comprising the voltage-sensing domain and the last two transmembrane segments (S5 to S6, in blue) the pore domain. The dotted cylinder in the S1–S2 linker is based on helical periodicity detected with alanine-scanning mutagenesis (Li-Smerin et al. 2000a). The top is extracellular and the bottom is intracellular. (B) Architecture of voltage-gated K+ channels containing a central pore domain and four surrounding voltage-sensing domains drawn according to Li-Smerin et al. 2000b. The pore domain is represented by the crystal structure of KcsA (Doyle et al. 1998), a simple K+ channel homologous to the S5–S6 region of voltage-gated K+ channels. Adjacent pore domain subunits in the tetramer are illustrated using different shades of blue. The red ring over the ion conduction pore indicates the footprint of Agitoxin2 (∼20 × 30 à ), a pore-blocking toxin (MacKinnon et al. 1998). Each surrounding voltage-sensing domain is formed by S1–S4 transmembrane helices. The four areas surrounded by black dotted lines are regions that potentially interact with Hanatoxin, based on cohabitation of Hanatoxin and Agitoxin2. The overall dimensions of the channel are drawn using estimates of 80 × 80 à from electron microscopy (Li et al. 1994).
	Figure 3. Effects of multiple substitutions at position 274 on Hanatoxin binding affinity. (A) Concentration dependence for inhibition of the wild-type and two mutant drk1 K+ channels by Hanatoxin. In/In0 is the value of I/I0 measured in the plateau phase at negative voltages (e.g., Fig. 2 C). Symbols are experimental data for the mean ± SEM of three to five cells. Solid lines correspond to In/In0 = (1 − P)4, where P = [toxin]/([toxin] + Kd), with Kd values of 103 nM, 4.4 μM, and 61.6 μM for WT, F274G, and F274R, respectively. The equation assumes four equivalent and independent toxin binding sites per channel. (B) Normalized Kd values for 14 substitutions at the position 274. Mean ± SEM (n = 3–15) for each mutant channel. The corresponding ΔΔG values are (kcal mol−1): 3.7 K, 3.6 R, 2.8 E, 2.6 S, 2.6 D, 2.4 G, 1.8 C, 1.6 A, 1.5 M, 1.5 P, 1.5 Y, 1.4 W, 1.4 V, and 1.2 I.
	Figure 4. Effects of multiple substitutions at position 277 on Hanatoxin binding affinity. (A) Concentration dependence for inhibition of the wild-type and two mutant drk1 K+ channels by Hanatoxin. In/In0 is the value of I/I0 measured in the plateau phase at negative voltages (e.g., Fig. 2 C). Symbols are experimental data for the mean ± SEM of three to six cells. Solid lines correspond to In/In0 = (1 − P)4, where P = [toxin]/([toxin] + Kd), with Kd values of 2.5 and 17.3 μM for E277Y and E277K, respectively. Data for the wild-type channel are the same as in Fig. 3. (B) Normalized Kd values of eight substitutions at position 277. Mean ± SEM (n = 3–13) for each mutant channel. The corresponding ΔΔG values are (kcal mol−1): 2.8 R, 2.8 K, 2.2 W, 2.0 Y, 1.6 G, 1.5 Q, 1.3 N, and 0.04 D.
	Figure 5. Effects of multiple substitutions at position 273 on Hanatoxin binding affinity. (A) Concentration dependence for inhibition of the wild-type and a mutant drk1 K+ channel by Hanatoxin. In/In0 is the value of I/I0 measured in the plateau phase at negative voltages (e.g., Fig. 2 C). Symbols are experimental data for the mean ± SEM of three to four cells. Solid lines correspond to In/In0 = (1 − P)4, where P = [toxin]/([toxin] + Kd), with a Kd value of 12.9 μM for I273Y. Data for the wild-type channel are the same as presented in Fig. 3. (B) Normalized Kd values of 13 substitutions at I273. Mean ± SEM (n = 3–10) for each mutant channel. The corresponding ΔΔG values are (kcal mol−1): 2.8 Y, 1.7 C, 1.6 G, 1.6 A, 1.6 D, 1.5 R, 1.5 T, 0.3 V, 0.3 M, 0.2 W, 0.1 F, 0.05 H, 0.03 L.
	Figure 6. Alanine-scanning mutagenesis of S1 to S3. Bar graph plot of normalized Hanatoxin Kd values for mutations spanning from K185 in S1 to T311 in S4. Data for Y270 in the COOH terminal of S3 through T311 in S4 are presented from a previous study (Swartz and MacKinnon 1997b) for comparison. Letters and numbers indicate the wild-type residues and their positions, respectively. All residues were mutated to Ala, except for the native Ala residues, which were mutated to Tyr. Kd values are mean ± SEM with n = 3 or 4 for each mutant obtained using between 100 and 250 nM Hanatoxin. The solid line superimposed on the bar graph is a 17-residue window analysis of the Kyte-Doolittle hydrophobicity index (Kyte and Doolittle 1982).
	Figure 7. Effects of the mutations in the peripheral surface of the pore domain. (A) Sequence alignment for the pore domain of the drk1 K+ channel and transmembrane part of KcsA. Numbers above and below the sequences indicate the positions of the residues for each channel. Yellow highlighting marks conserved residues between the two K+ channels. Cylinders indicate the demarcations of the two transmembrane α helices (TM1 and TM2 for KcsA and S5 and S6 for the drk1 K+ channel) and the pore helix. *18 positions in the drk1 K+ channel where mutations were made and the effects on Hanatoxin binding affinity were determined. All residues were mutated to Ala except A348L, W365F, and L384F. (B) Location of KcsA residues (shown in gray) that correspond to those mutated in the drk1 K+ channel. Gray residues correspond to those marked by asterisks in A. The crystal structure of the tetrameric KcsA channel shown for a top view (left) or a side view (right). Numbers correspond to residues in the drk1 K+ channel. Yellow dotted rings show two orientations of the Agitoxin2 footprint. (C) Normalized Kd values for 18 mutations in the pore domain of the drk1 K+ channel. Data are mean ± SEM with n = 3 or 4 for each mutant channel. Dashed line marks a normalized Kd value of 1.
	Figure 8. Molecular determinants and localization of the Hanatoxin receptors on the drk1 K+ channel. (A) Surface rendering for one face of Hanatoxin1. The structure is a solution structure solved by NMR spectroscopy (Takahashi et al. 2000). The cluster of hydrophobic residues (green) surrounded by basic (blue) and acid (red) residues represents a surface feature common to Hanatoxin1, CsEV (an α-scorpion toxin) and ATXIII (a sea anemone toxin). (B) Putative topology and secondary structure of voltage-gated K+ channel α subunits. The first four transmembrane segments (S1–S4, in black) comprise the voltage-sensing domain and the last two transmembrane segments (S5–S6, in blue) in the pore domain. See Fig. 1 legend for details of secondary structure. The top is extracellular and the bottom is intracellular. A short stretch of sequence is shown for the COOH-terminal part of S3, beginning with the P268 (bold), a residue that is conserved in voltage-gated K+ channels. Red lettering indicates the positions of the three residues (I273, F274, and E277) that interact intimately with Hanatoxin. (C) Architecture of a voltage-gated K+ channel containing a central pore domain and four surrounding voltage-sensing domains drawn according to Li-Smerin et al. 2000b. Black circles shown for one voltage-sensing domain illustrate the four transmembrane (S1–S4) helices contained within, positioned according to Li-Smerin et al. 2000a. Yellow patches illustrate the Hanatoxin receptors confined within the voltage-sensing domains.

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