Kitaguchi T , Sukhareva M , Swartz KJ .

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

	Figure 1. . The intracellular gate region of K+ channels. (A) Cartoon of a voltage-gated K+ channel showing separate voltage-sensing and pore domains and the S6 activation gate toward the intracellular side of the pore. (B) Membrane folding diagram for a Kv channel showing six TM domains. S1–S4 comprise the voltage-sensing domains, while S5–S6 forms the pore domain. Black circle in pore region illustrates the position of W434 and green circle in activation gate region illustrates the position of V478. (C) Sequence alignment between Kv channels, KcsA, MthK, and KirBac. Red highlighting marks the conserved Gly residue that is proposed to serve as the gating hinge (Jiang et al., 2002a,b). Blue highlighting marks the position of P475 in Shaker, corresponding to P406 in Kv2.1, G229 in KvAP, A108 in KcsA, E92 in MthK, and G143 in KirBac. Green highlighting marks positions equivalent to V478 in Shaker, where W substitutions result in nonconducting channels (Hackos et al., 2002).
	Figure 2. . Gating currents and Q–V relations for two Shaker Kv channel mutants that display a nonconducting phenotype. (A) Families of gating currents for two mutant Shaker K+ channels. For both families, holding voltage was −100 mV and depolarizations were to voltage between −100 mV and 0 mV with 10-mV increments. A P/−4 protocol was used to subtract leak and linear capacitive currents. (B) Normalized Q–V relations for W434F and V478W. Q was obtained by integrating both ON and OFF components of gating current, taking their average and normalized to Qmax measured at depolarized voltage. Data points are mean ± SEM. Smooth curves correspond to single Boltzmann functions with parameters as follows: W434F, V50 = −47.6 mV, z = 3.6; V478W, V50 = −47.3 mV, z = 3.2. See Table I for statistics.
	Figure 3. . Effects of the T449V mutation on two nonconducting mutations in the Shaker Kv channel. Families of ionic and gating currents are shown for W434F and V478W, with and without the T449V mutation. For gating current families, holding voltage was −100 mV and depolarizations were from −100 to 0 mV, in 10-mV increments. For W434F+T449V, holding voltage was −100 mV, tail voltage was −100 mV, and depolarizations were from −100 to 40 mV, in 10-mV increments. A P/−4 protocol was used to subtract leak and linear capacitive currents.
	Figure 4. . Ionic and gating currents for dimeric Shaker constructs. (A) Schematic diagram illustrating dimeric channel constructs. A and B protomers are concatenated using 10-residue linkers. (B) Families of ionic and gating currents for homodimers of Wt and V478W-containing subunits. For Wt–Wt, holding voltage is −100 mV, tail voltage was −60 mV, and depolarizations were from −60 to 40 mV, in 10-mV increments. For V478W–V478W, holding voltage was −100 mV and depolarizations were from −100 to 0 mV, in 10-mV increments. (C) Families of ionic and gating currents for the V478W–Wt heterodimer. Holding voltage was −100 mV and depolarizations were from −100 to 50 mV, in 10-mV increments, with or without 1 μM Agitoxin-2. A P/−4 protocol was used to subtract leak and linear capacitive currents. All constructs were recorded using an external solution containing 100 mM KCl. (D) G–V relations for Wt–Wt and V478W–Wt dimers. For Wt–Wt, normalized tail current amplitudes, measured at voltages indicated in B, are plotted versus the voltage of the preceding depolarization. For V478W–Wt, the ionic tail currents are significantly contaminated by OFF gating current and therefore conductance was calculated from the amplitude of steady-state current before repolarization, normalized to the value at +100 mV and plotted versus voltage. Agitoxin-2 was used to isolate ionic currents from endogenous currents. Data points are mean ± SEM. Smooth curves are single Boltzmann fits to the data with parameters as follows: Wt–Wt, V50 = −27 mV, z = 3.4; V478W–Wt, V50 = +7 mV, z = 1. (E) Normalized Q–V relations for V478W–V478W and V478W–Wt dimers. Q was obtained by integrating both ON and OFF components of gating current, taking their average and normalized to Qmax measured following strong depolarizations. Data points are mean ± SEM. Smooth curves are single Boltzmann fits to the data with parameters as follows: V478W–V478W, V50 = −51 mV, z = 3.2; V478W–Wt, V50 = −45 mV, z = 2.2. See Table I for statistics.
	Figure 5. . Inactivation kinetics for W434F and V478W dimers. (A) Current records for long test pulses to +40 mV. All constructs were recorded using an external solution containing 100 mM KCl. (B) Inactivation time constants for dimer constructs. Single exponential functions were fit to inactivating currents for Wt–Wt (n = 12), V478W–Wt (n = 6), and Wt–V478W (n = 14), while double exponential functions were fit to inactivating currents for W434F–Wt (n = 10) and Wt–W434F (n = 12) dimers. Data points are mean ± SEM.
	Figure 6. . Effects of internal TEA on gating charge immobilization in two Shaker Kv channel mutants. (A) Families of gating currents for two Shaker mutants in the absence and presence of internal TEA. For all mutants, holding voltage was −100 mV and depolarizations were from −100 to 0 mV, in 10-mV increments. A P/−4 protocol was used to subtract leak and linear capacitive currents. (B) Plots of τQoff against test voltage in the absence or presence of internal TEA. τQoff was obtained by fitting single exponential functions to the time course of OFF gating currents. For W434F in the presence of TEA, following test depolarizations to −50 mV, a double exponential function was fit to the OFF gating current. Data points are mean ± SEM. n = 3 for V478W and n = 4 for W434F.
	Figure 7. . Gating currents and Q–V relations for Shaker Kv channels with aromatic substitutions at V478. (A) Families of gating currents for three mutant Shaker Kv channels. For all families, holding voltage was −100 mV and depolarizations were to voltage between −100 mV and 0 mV with 10-mV increments. A P/−4 protocol was used to subtract leak and linear capacitive currents. (B) Normalized Q–V relations. Q was obtained by integrating both ON and OFF components of gating current, taking their average and normalized to Qmax measured following strong depolarizations. Smooth curves are single Boltzmann fits to the data with parameters as follows: V478W, V50 = −48 mV, z = 3.3; V478F, V50 = −54 mV, z = 3.3; V478Y, V50 = −66 mV, z = 2.9. See Table I for statistics.
	Figure 8. . Gating properties of Shaker V478 mutants displaying weakly conducting phenotypes. (A) Family of ionic and gating currents for the V478I mutant. Holding voltage was −100 mV and depolarizations were from −90 to 30 mV, in 10-mV increments. (B) Family of ionic and gating current for the V478L mutant. Holding voltage was −100 mV and depolarizations were from −90 to 80 mV, in 10-mV increments. (C) Families of ionic and gating current for the V478M mutant in the absence or presence of 1 μM Agitoxin-2. Holding voltage was −100 mV and depolarizations were from −100 to 100 mV, in 10-mV increments. (D) Family of ionic and gating current for the V478G mutant in the absence or presence of 1 μM Agitoxin-2. Holding voltage was −100 mV and depolarizations were from −100 to 100 mV, in 10-mV increments. (E) G–V relations for three weakly conducting mutants. Conductance was calculated from the amplitude of steady-state current before repolarization, normalized to Gmax estimated from Boltzmann fits and plotted versus voltage. Agitoxin-2 was used to isolate ionic currents from endogenous currents. Data points are mean ± SEM. Smooth curves are single Boltzmann fits to the data with parameters as follows: V478I, V50 = +88 mV, z = 1; V478L, V50 = +80 mV, z = 0.8; V478M, V50 = +89 mV, z = 1. (F) Normalized Q–V relations for three weakly conducting mutants. Q was obtained by integrating both ON and OFF components of gating current, taking their average and normalized to Qmax measured for strong depolarizations. Data points are mean ± SEM. Smooth curves are single Boltzmann fits to the data with parameters as follows: V478I, V50 = −56 mV, z = 2.8; V478L, V50 = −49 mV, z = 2.3; V478M, V50 = −41 mV, z = 2.1; V478G, V50 = −42 mV, z = 3.1; V478W, V50 = −48 mV, z = 3.3. In all instances a P/−4 protocol was used to subtract leak and linear capacitive currents. See Table I for statistics.
	Figure 9. . Gating properties of Shaker V478 mutants displaying a normal conducting phenotype. (A) Families of ionic currents for Wt Shaker and three normal conducting mutants. For Wt and V478A, holding voltage was −100 mV, tail voltage was −60 mV, and depolarizations were from −60 to 40 mV, in 10-mV increments. For V478Q, holding voltage was −100 mV, tail voltage was −100 mV, and depolarizations were from −90 to 20 mV, in 10-mV increments. For V478S, holding voltage was −100 mV, tail voltage was −50 mV, and depolarizations were from −50 to 50 mV, in 10-mV increments. A P/−4 protocol was used to subtract leak and linear capacitive currents. (B) G–V relations for Wt Shaker and three normal conducting mutants. In all cases, normalized tail current amplitudes, measured at voltages indicated in A, are plotted versus the voltage of the preceding depolarization. Data points are mean ± SEM. Smooth curves are single Boltzmann fits to the data with parameters as follows: Wt, V50 = −30 mV, z = 3.8; V478A, V50 = −40 mV, z = 3.6; V478Q, V50 = −37 mV, z = 5.5; V478S, V50 = −28 mV, z = 3. See Table I for statistics.
	Figure 10. . Nonexpressing mutants of the Shaker Kv channel and summary of phenotypes resulting from substitutions at V478. (A) Western blot from SDS polyacrylamide gel of c-myc–tagged Shaker protein obtained from oocyte membrane preparations. Each lane contains three oocyte equivalents of Shaker protein. Wt protein has two dominant forms, a core-glycosylated species (band ∼70 kD) and a more heavily glycosylated mature form (band at ∼100 kD). The N259Q/N263Q double mutant shows only a single band at ∼65 kD and marks the position of the unglycosylated protein. (B) Summary of phenotypes resulting from different substitutions at V478.
	Figure 11. . Rescue of ion conduction in V478W by secondary mutations at P475. (A) Families of gating and ionic current for V478W, P475Q, and the double mutant. For V478W, holding voltage was −100 mV, depolarizations were from −100 to 0 mV (10-mV increments) and a P/−4 protocol was used to subtract leak and linear capacitive currents. For P475Q, holding and tail voltages were −150 mV and depolarizations were from −150 to 30 mV, in 10-mV increments. For V478W/P475Q, holding and tail voltages were −100 mV and depolarizations were from −90 to 40 mV, in 10-mV increments. For both P475Q and V478W/P475Q, leak and linear capacitive currents were identified and subtracted by blocking the Shaker channel with Agitoxin-2. (B) G–V relations for Wt, two P475 mutants displaying constitutive activity and two double mutants displaying normal voltage-dependent gating. Normalized tail current amplitudes are plotted versus the voltage of the preceding depolarization. For P475D, the external solution contained 50 mM KCl, whereas for all other constructs the external solution contained 50 mM RbCl. Data points are mean ± SEM. Smooth curves are single Boltzmann fits to the data with parameters as follow: Wt, V50 = −30 mV, z = 3.8; P475Q, V50 = −97 mV, z = 2.6; P475D, V50 = −105 mV, z = 2.8; V478W/P475Q, V50 = −45 mV, z = 2.1; V478W/P475D, V50 = −17 mV, z = 3. See Table I for statistics.
	Figure 12. . Single channel properties of Wt Shaker and the V478W–Wt dimer channel recorded in symmetrical K+. (A) Representative current traces recorded from Wt Shaker (left) and V478W–Wt dimer (right) in symmetrical 140 mM KCl. The single channel patch was held at −100 mV and stepped from −100 mV to +40 mV with 20-mV increments for 400 ms and returned back to −100 mV. The traces represent segments of current records during the test pulse where single channel current events are observed. Filter frequency was 2 kHz. Horizontal dashed red and blue lines correspond to the closed and open current levels, respectively. (B) I–V relationship for the open state of the Wt Shaker channel. Linear regression of the shown data yields a conductance of 24 pS. (C) Open probability for the highest conducting state as a function of voltage for Wt (open circles) and for V478W–Wt dimer (filled circles). Smooth curve for Wt is a fit of a single Boltzmann function to the data with V50 = −54 mV and z = 2.5. Each data point represents the mean ± SEM (n = 5–6).
	Figure 13. . Single channel properties of the Shaker P475Q and V478W+P475Q mutant channel. (A) Representative current traces recorded for P475Q (left) and V478W+P475Q (right) in symmetrical 140 mM KCl. The single channel patch was held at −100 mV and stepped from −100 mV to 0 mV with 20-mV increments for 400 ms and returned back to −100 mV. The traces represent segments from current records where single channel current events were observed. Filter frequency was 2 kHz. Horizontal dashed red and blue lines correspond to the closed and open current levels for single mutant, respectively.
	(SCHEME 1).
	Figure 14. . Pore structures of four K+ channels viewed from an intracellular vantage point. In all instances, residues at the position equivalent to V478 are shown as CPK with carbon colored gray, oxygen colored red, and nitrogen colored blue. See Fig. 1 for sequence alignment. Residues at the equivalent position to P475 are shown in CPK with all atoms colored light blue. PDB accession code for KcsA is 1J95. PDB coordinates for KirBac provided by D.A. Doyle (University of Oxford, Oxford, UK). PDB accession code for MthK is 1LNQ. For MthK, E92 was modeled as an Ala. PDB accession code for KvAP is 1ORQ. Structures were generated using DS Viewer Pro (Accelrys).

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