Pathak M , Kurtz L , Tombola F , Isacoff E .

R01 NS035549-08 NINDS NIH HHS , R01 NS035549 NINDS NIH HHS

	Figure 1. . The ILT mutant channel. (A) The S4 sequence for Shaker and for the ILT mutant channel. Charged residues are denoted in bold and numbered. (B) Cartoon illustrating activation and opening in WT and ILT channels. Only two subunits of the channel are shown, with the positively charged helix S4 and the S6 helix. The long gray arrow represents a voltage axis ranging from −150 to 200 mV. In the WT channel (top), activation and opening are tightly coupled and the activated state is short-lived (denoted by a bracket around the activated state). In the ILT channel (bottom), activation is negatively shifted, whereas opening is positively shifted. Cartoon based on results from Ledwell and Aldrich (1999).
	Figure 2. . S6 gate in ILT channels is closed in the activated conformation. (A) Cartoon showing that the NH2-terminal ball can block the channel only when the S6 gate is open. (B) ILT+ball channels show fast inactivation in excised inside-out macropatches. Inset shows voltage protocol used, HP = −100 mV. (C) Fast inactivation in ILT+ball channels takes place over the voltage range of opening, not activation. Steady-state (S.S) prepulse fast inactivation for ILT+ball channels (closed circles) measured from peak amplitude of current during a 20-ms test pulse to +180 mV following a 100-ms voltage step to the specified potential, HP = −100 mV with a 50-ms prestep to −120 mV. G–V from ILT channels (open circles) constructed as described in materials and methods, Q–V (open squares) measured from integrated OFF gating currents of ILT W434F channels. Solid lines are Boltzmann fits to the data. Fit parameters: ILT Q–V, V1/2 = −70.4 mV, slope factor = 16.3 mV, representative of n = 8; ILT+ball prepulse inactivation curve, V1/2 = +77.3 mV, slope factor = 13.3, average of data from 11 patches; ILT G–V, V1/2 = +91.8 mV, slope factor = 14.9, average of data from 8 patches. (D) Two voltage pulses to +180 mV, from a prestep to −120 mV and from an ensuing prestep to 0 mV, yield currents with similar amplitude in ILT+ball channels. Representative trace of n = 6.
	Figure 3. . S4 attached TMRM senses protein motion over voltage range of ILT channel opening. (A) Normalized steady-state F–V for 359C WT W434F (open diamonds) and for 359C ILT W434F (closed circles). F–Vs are normalized to the amplitude of single Boltzmann fit for 359C WT and a double Boltzmann fit for 359C ILT (fits are solid lines). Fit parameters: 359C WT W434F V1/2 = −43.8 mV, slope factor = 7.3, representative of n = 13; 359C ILT W434F V11/2 = −66.8 mV, slope factor = 17.2, V21/2 = +105.6 mV, slope factor = 24.1, representative of n = 18. Traces on top show the fluorescence intensity change evoked from a prestep of −120 mV to voltage step to 0 mV (gray) or 180 mV (black) for 359C ILT W434F channels. HP = −80 mV. (B and C) Superimposition of the Q–V from 359C-TMRM ILT W434F channels (B, filled circles), and the G–V from 359C-TMRM ILT channels (C, filled circles) with the F–V from 359C-TMRM ILT W434F channels (open circles). F–V and Q–V measured in two-electrode voltage clamp configuration, and G–V measured from excised inside-out macropatches. Each data point is a mean of n = 5 for the Q–V, n = 9 for G–V, and n = 9 for F–V. Solid lines are Boltzmann fits to the data. Fit parameters: Q–V V1/2 = −67.3 mV, slope factor = 25.8; G–V V1/2 = 94.3 mV, slope factor = 22; F–V double Boltzmann fit parameters: V11/2 = −69.5 mV, slope factor = 17.6, V21/2 = +105.1 mV, slope factor = 23.3.
	Figure 4. . Opening rearrangement sensed at four sites in S4 and proximal S3–S4 linker. Steady-state fluorescence behavior of TMRM attached to sites 351C (A), 356C (B), 358C (C), and 361C (D). Each panel shows the F–Vs for TMRM attached to the specified site in the WT W434F background (open diamonds) and in the ILT W434F background (filled circles). Open circles show G–Vs measured from excised inside-out macropatches for ILT channels labeled with TMRM at the specified site. Traces on top of each graph show the fluorescence intensity change in response to a step to 0 mV (gray) and 180 mV (black) from a prestep to −120 mV in the ILT W434F background. HP = −80 mV. Different amounts of separation between the WT and ILT F–Vs at the four positions likely reflect different impacts on gating of the cysteine mutation and TMRM labeling. F–Vs are representative of n = 7 for 351C ILT, n = 6 for 351C WT, n = 3 for 356C ILT, n = 10 for 356C WT, n = 3 for 358C ILT, n = 6 for 358C WT, n = 6 for 361C ILT, n = 4 for 361C WT. Fit parameters for G–Vs: 351C ILT V1/2 = 113.6 mV, slope factor = 21.2, n = 3; 356C ILT V1/2 = 96.1 mV, slope factor = 20.5, n = 6; 358C ILT V1/2 = 94.3 mV, slope factor = 22.0 mV, n = 6; 361C ILT V1/2 = 102.9 mV, slope factor = 19.5, n = 9.
	Figure 5. . 4-AP eliminates the fluorescence change over the voltage range of opening for 359C-TMRM ILT. (A) Fluorescence changes for 359-TMRM ILT W434F channels from the same cell in the voltage range of opening in the absence (left) and presence (right) of 2 mM 4-AP added to the bath solution. Voltage protocol is shown as inset. (B) Steady-state fluorescence plotted as a function of voltage for channels in the absence of 4-AP (open circles) and in the presence of 2mM 4-AP (filled circles).
	Figure 6. . Slow inactivation in ILT channels over voltage range of S4's gating motion. (A, i) Superimposed currents in response to a 1-s depolarizing step to +150 mV for ILT channels (gray trace) and ILT/T449V channels (black trace) show that the T449V mutation slows slow inactivation of ILT channels. HP = −100 mV, post-step voltage = +40 mV. (ii) Voltage protocol (left, bottom) and currents (left, top) used for measuring the voltage dependence of slow (P-type) inactivation from conducting ILT channels in excised inside-out macropatches. Interval between sweeps = 5 s at an HP of −100 mV. The fraction of current after inactivation (FCAI, right, filled symbols) was plotted from the amplitude of the test pulse following the 1-s inactivating pulses after correcting for run-down in ionic current from patch (see materials and methods). G–V for ILT channels measured from the same patch (right, open symbols). Data are representative of n = 3 patches. Solid lines are Boltzmann fits to the data. Fit parameters for data from three patches: for ILT Slow inactivation, V1/2 = +108.6 ± 9.8 mV; G–V, V1/2 = +101.4 ± 6.9 mV. (B) C-type inactivation does not occur from the activated state. Representative F–Vs for 359C WT W434F (left,) and 359C ILT W434F (right), from an HP of −80 mV (open symbols) or 0 mV (closed symbols). Interval between traces = 9 s. Solid lines represent single Boltzmann fits for WT and double Boltzmann fits for ILT. For 359C WT W434F, representative of n = 3: −80 mV hold, V1/2 = −41.4 mV, slope factor = 9.3; 0 mV hold, V1/2 = −48.9 mV, slope factor = 8.3. For 359C ILT W434F, representative of n = 4: double Boltzmann fit with −80 mV hold, V11/2 = −55.9 mV, slope factor 1 = 14.0, V21/2 = +111.4 mV, slope factor 2 = 20.5; 0 mV hold, V11/2 = −58.6 mV, slope factor 1 = 14.1, V21/2 = +111.1 mV, slope factor 2 = 23. (C) ILT channels undergo C-type inactivation. (i) Tail currents following a voltage step to 0 mV for 449V WT (left) and 470C/449V WT (right) channels. Voltage protocol is shown as inset; ta is the duration of voltage step = 10, 40, 100, 400, 1,000, or 1,400 ms. (ii) Tail currents for 449V ILT and 470C/449V ILT channels following a voltage step to 150 mV with ta = 20, 100, 200, 400, 1,000, or 1,500 ms. Recordings are from excised inside-out macropatches in symmetric (140 mM K+) conditions. Tail current voltage for ILT channels is +40 mV since the inward tails at −80 mV are fast and hard to distinguish from the capacitive transient.
	Figure 7. . S4s move cooperatively over the voltage range that drives gating. (A) Subunit stoichiometry for hetero-tetrameric channels and their controls. * indicates a labeled subunit. Symbols shown under cartoons are same as used in graphs B and C. (B) Mean of F–Vs from labeled ILT*(WT)3 (filled circles), (WT*)4 (open, inverted triangles), and (ILT*)4 channels (open circles). Solid lines are Boltzmann fits to the data. Fit parameters: ILT*(WT)3 V1/2 = −50.9 mV, slope factor = 19, n = 6; (WT*)4 V1/2 = −39.1 mV, slope factor = 8.2, n = 4. For (ILT*)4, double Boltzmann fit parameters: V11/2 = −69.5 mV, slope factor = 17.6, V21/2 = +105.1 mV, slope factor = 23.3, n = 11. (C) Mean of F–Vs from labeled WT*(ILT)3 (filled, inverted triangles). Fit parameters for WT*(ILT)3 double Boltzmann fit V11/2 = −35.7 mV, slope factor = 12.0, V21/2 = 28.3 mV, slope factor = 20.5, n = 11; fits parameters to (WT*)4 and (ILT*)4 channels are same as in B.
	Figure 8. . A model for channel opening. (A) Mapping of Shaker ILT positions onto the crystal structure of KvAP's isolated voltage sensing (S1–S4) domain (Jiang et al., 2003a) shows ILT residues to face away from other membrane segments in direction deduced to interact with the pore domain in the activated conformation (Gandhi et al., 2003). ILT residues are colored. P.D., pore domain. (B) Model for association between activation and gating motions of S4 and opening of the S6 gate. Top layer shows a side view of two subunits of the channel; dotted lines point to a top view of the channel with all four subunits shown. The S4s (orange cylinders) move independently (or with a mild cooperativity) of each other during activation, and do not exert force on the S6 gate (green cylinders). In the activated state, each S4 interacts with its neighboring S5 (blue oval, top view), which is denoted by dashed line. From the activated state, the S4s undergo a cooperative motion in which each S4 places strain on the S6 gate via S4–S5 (hook and spring representation). The interaction of an S4 with its neighboring S5 is stronger in ILT, hence a strong depolarization is needed to release S4 from its activated conformation to undergo its final motion.

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