Xu Y , Ramu Y , Shin HG , Yamakaze J , Lu Z .

GM55560 NIGMS NIH HHS , R01 GM055560 NIGMS NIH HHS , Howard Hughes Medical Institute

	Figure 2. Deletion analysis of CTS3. (a) CTS3 and neighboring sequences of wild-type and mutant channels. Mutant channels containing 0–10 tryptophan residues in CTS3 are denoted as 0W–10W. Asterisks signify that the EEED sequence was also deleted. (b–o) Currents of wild-type and mutant channels, elicited by stepping membrane voltage from the −100 mV holding potential to between −100 mV and 80 mV (b) or 110 mV (c–o) in 10 mV increments. Currents in c were corrected for background currents obtained with 1 µM agitoxin-1 (AgTx1) present.
	Figure 3. Stepwise deletions in CTS3 and the S3–S4 linker. (a) Sequence of CTS3 through NTS4. (b–g) Currents of mutant channels that lack the entire CTS3 and partial S3–S4 linker sequences as indicated. Currents were elicited by stepping membrane voltage from the −100 mV holding potential to between −80 mV and 80 mV in 10 mV increments. Current shown in b, d and e were corrected for background currents with the P/4 protocol. (h) G-V curves of the deletion mutants, along with that of wild-type (Fig. 1b), where the curves are fits of a Boltzmann function, yielding V1/2 = 6 ± 1.2 mV and Z = 2.5 ± 0.2 (mean ± s.e.m., n = 10) for ΔY323–P341; V1/2 = 35 ± 0.5 mV and Z = 2.0 ± 0.1 (n = 14) for ΔY323–K342; V1/2 = 3.0 ± 0.7 mV and Z = 3.5 ± 0.3 (n = 8) for ΔY323–A343; and V1/2 = 22 ± 0.7 mV and Z = 1.6 ± 0.1 (n = 6) for ΔY323–P344. We calculated conductance values for the G-V curve of ΔY323–K342 from the current and K+-driving force ratio, and used the tail current method for the other three mutants.
	Figure 4. Deletion analysis of CTS3 through NTS4. (a–c) Currents of mutant channels elicited in the presence of 20 mM (a) or 100 mM (b and c) extracellular K+ by stepping membrane voltage from the −80 mV (a) or 0 mV (b and c) holding potential to between −70 mV (a) mV or −120 mV (b and c) and 80 mV in 10 mV increments. In the mutant channels, the sequences from I325 to V367 (a), F324 (b) or Y323 (c) were deleted and replaced by a glycine triplet. Currents shown were corrected for background currents obtained with 1 µM agitoxin-1 (AgTx1) present.
	Figure 5. Cysteine point mutations of CTS3 in the presence of a hexa-cysteine mutation in NTS4. (a) Sequences of CTS3 and NTS4 without or with a hexa-cysteine mutation. (b–g) Current traces of mutant channels elicited by stepping from the −100 mV holding potential to between −80 mV and 80 mV in 10 mV increments. All six mutants contain a hexa-cysteine mutation as shown in a, without (b) or with (c–g) additional cysteine mutation in CTS3, as indicated. Currents shown in d were corrected for background currents obtained with 1 µM AgTx1 present.
	Figure 6. Cysteine mutation of individual hydrophobic residues in NTS4 in the presence of I325C in CTS3. Current traces of mutant channels elicited by stepping from the −100 mV holding potential to between −80 mV and 80 mV in 10 mV increments. All six mutants contain the I325C mutation in CTS3 and an additional cysteine mutation in NTS4, as indicated. Currents of the I325C I364C double mutant were corrected for background currents obtained with 1 µM AgTx1 present.
	Figure 7. Cysteine pairs between CTS3 and NTS4 that lock the channels in the open state. (a) Sequences of CTS3 and NTS4 of Shaker and Kv1.2–2.1 channels. The residue pairs in the Shaker sequence, whose substitution by cysteine lock the channel in the open state, are colored lime and blue. Corresponding residues in the Kv1.2–2.1 sequence are similarly colored. (b) Structure of Kv1.2–2.1’s CTS3 through NTS4 (PDB: 2R9R). Colored sticks correspond to the colored residues in the Kv1.2–2.1 sequence in a. (c–f) Ionic currents of the I325C I364C (c and d) or T329C L361C (e and f) double mutant without (control) or with exposure to 1 mM DTT (d, a few minutes; f, overnight). Currents were elicited by stepping membrane voltage from −100 mV (c and e) or −120 mV (d and f) to 100 mV (c and e) or 50 mV (d and f) in 10 mV increments. Traces shown in c and e were corrected for background currents obtained with 1 µM AgTx1 present. (g–j) Gating currents of channels containing the W434F mutation and the I325C I364C (g and h) or T329C L361C (i and j) double mutation without (control) or with exposure to 1 mM DTT (h, a few minutes; j, overnight). Currents elicited by stepping membrane voltage from −140 mV to 0 mV in 10 mV increments. Bathing solutions contained 100 mM K+ (c–f) or 5 mM K+ plus 95 mM Na+ (g–j).
	Figure 8. Biochemical examination of disulfide bond formation between cysteine pairs in the paddle motif. (a and b) Western blots of purified recombinant wild-type Shaker protein and I325C I364C (a) or (b) T329C L361C double-cysteine mutant proteins prepared under reducing or non-reducing conditions and with or without TEV digestion. All tested proteins contain an N-terminal Flag epitope with or without a TEV site in the S3–S4 linker. Molecular weight standards (MWS) run in the leftmost lane.
	Figure 9. Partial structures of Kv1.2–2.1. (a) Space filling model of S3–S5 where hydrophobic, polar, negatively charged and positively charged residues are colored orange, magenta, ruby, and blue, respectively (PDB: 2R9R). S4 is delineated in turquoise. (b) Model of S1–S6, with S1–S4 from one subunit and S5 and S6 from the adjacent subunit. S3–S5 are positioned and colored as in a, whereas S1, S2 and S6 are shown as cyan, light blue, and lime ribbons, respectively. (c and d) Back views of a and b, respectively. The dotted lines approximate the membrane boundaries, extracellular (EC) and intracellular (IC) sides above and below, respectively.

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