Darman RB , Ivy AA , Ketty V , Blaustein RO .

R01HL68985 NHLBI NIH HHS , T32HL069770 NHLBI NIH HHS , R01 HL068985 NHLBI NIH HHS , T32 HL069770 NHLBI NIH HHS

	(SCHEME 1).
	Figure 1. (A) Transmembrane topology of a Shaker K+ channel subunit highlighting residues discussed in the text. (B) Alignment of Shaker and KvAP from Jiang et al. (2003a) showing the difference in length of the two S3–S4 linkers. Residues discussed in the text are in bold; arrows indicate portion of Shaker's S3–S4 loop removed via mutagenesis. (C) Chemical structure of maleimido-Gly7TEA (abbreviated as Gly7TEA).
	Figure 2. Normalized Q-V relationships for WT Shaker (circles; n = 3 oocytes) and Shaker E334C (squares; n = 4 oocytes). Qnorm was determined as described in the text. Data points are mean ± SEM. Smooth curves are fits to single Boltzmann functions with V1/2 and z values of −36.5 mV and 2.5 for WT, and −38.2 mV and 2.8 for E334C.
	Figure 3. Kinetics of Gly7TEA tethering to D336C. (A) Templates of the pulse protocols used to assay for state-dependent tethering. In each case, oocytes are depolarized to +20 mV and hyperpolarized to −110 mV. Top, “short-pulse” protocol in which 50-ms depolarizations are applied every 5 s. Bottom, “long-pulse” protocol in which 1-s depolarizations are applied every 3 s. (B) Top, currents in response to short-pulse protocol. Upper trace is before addition of 50 μM Gly7TEA; bottom trace is after ∼23 min of exposure. Bottom, currents in response to long-pulse protocol with top and bottom traces as described above. (C) Kinetics of tethered block. Isochronal currents taken 1 ms before end of each pulse were normalized to their values before Gly7TEA exposure and plotted against time (short-pulse, circles; long pulse, triangles). Solid gray curves through the data are fits to double-exponential functions with taus (and weights) of 25 s (70%) and 212 s (30%) for circles, and 26 s (66%) and 190 s (34%) for triangles.
	Figure 4. Kinetics of Gly7TEA tethering to E334C Δ336–357. (A) Pulse protocols are as described in Fig. 3, except that the short-pulse protocol in these experiments employed a 100-ms depolarizing pulse to allow full activation since this construct exhibits slower activation kinetics. (B) Top, currents in response to short-pulse protocol. Top trace is before addition of 100 μM Gly7TEA; bottom trace is after ∼24 min of exposure. Bottom, currents in response to long pulse protocol with top and bottom traces as described above. (C) Kinetics of tethered block. Time courses were generated as described in Fig. 3 (short-pulse, circles; long pulse, triangles). Solid gray curves through the data are fits to double-exponential functions with taus (and weights) of 29 s (55%) and 303 s (45%) for circles, and 33 s (56%) and 318 s (44%) for triangles.
	Figure 5. Kinetics of Gly7TEA tethering to A359C. (A) Pulse protocols are as described in Fig. 3. (B) Top, currents in response to short-pulse protocol. Top trace is before addition of 400 μM Gly7TEA; bottom trace is after ∼78 min of exposure. Bottom, currents in response to long-pulse protocol. Top trace is before addition of 400 μM Gly7TEA; bottom trace is after ∼35 min of exposure. (C) Kinetics of tethered block. Time courses were generated as described in Fig. 3 (short-pulse, circles; long pulse, triangles). Solid gray curves through the data are fits to double-exponential functions with taus (and weights) of 50 s (58%) and 254 s (42%) for triangles, and 122 s (23%) and 999 s (77%) for circles.
	Figure 6. Kinetics of inhibition of biotinylated D336C channels by streptavidin. (A) Pulse protocols are as described in Fig. 4, except that depolarizations are every 10 s to allow for recovery from inactivation, and the hyperpolarized voltage was −90 mV in each case. (B) Currents through channels that have been biotinylated with MTSEA-biotin. Top traces in each set are before addition of 3.3 μM streptavidin; bottom traces are after ∼25 min of exposure. (C) Kinetics of inhibition. Isochronal currents at 99 ms were normalized to the values before streptavidin exposure, averaged from several experiments, and plotted against time. Error bars are ± SEM. Short-pulse protocol (n = 6), circles; long-pulse protocol (n = 8), triangles. Control experiments (three upper time courses) generated using the long-pulse protocol: diamonds, D336C with 1 mM MTSEA-biotin added to chamber; inverted triangles, streptavidin applied to D336 channels; squares, streptavidin applied to D336C channels that were prereacted with 1 mM MTS-glucose. (D) Biotin quenching using long-pulse protocols. 500 μM d-biotin added 60 s before addition of streptavidin (diamonds), and at 30 (squares), 60 (pentagons), and 120 s (circles) after streptavidin exposure. These time courses are from isochronal currents at 999 ms and are superimposed on the long-pulse data (triangles) obtained from the same experiments as in (C) except isochronal currents at 999 ms are plotted.
	Figure 7. Kinetic simulations of a 5-à movement of the top of S3 toward (A–C) or away (D–F) from the pore during depolarization. Movement of S3 along the surface of a hemisphere 35 à from the pore, depicted as a solid circular arc in A and D, would preserve radial distance. An inward movement during depolarization would involve a displacement of S3 to the surface of a hemisphere at 30 à , depicted as a dotted circular arc in A, and an outward movement would involve a displacement to the dotted hemisphere at 40 à in D. (B and E) Templates of pulse protocols used for the simulations. Upper protocols have 50-ms depolarizations followed by 4.95-s hyperpolarizations; bottom protocols are 1-s depolarizations followed by 2-s hyperpolarizations. k30 represents a set of four rate constants determined at 30 à as described in the text; k35 and k40 are similarly determined at 35 and 40 à , respectively. (C and F) Simulated time courses. Black curves are generated by application of short-pulse protocols; gray curves from long-pulse protocols. Each time course is generated by switching back and forth between the two sets of rate constants depicted in each protocol template in B and E.

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