Gagnon DG , Bezanilla F .

GM030376 NIGMS NIH HHS , R01 GM030376 NIGMS NIH HHS , R37 GM030376 NIGMS NIH HHS

	Figure 1. Functional effect of the stoichiometry of ILT subunits on Shaker. (A) Typical recordings of family of ionic currents from oocytes expressing heterotetramer constructs. The pulses were applied from an HP of −90 mV (with or without prepulse to −120 mV, as noted) to membrane potentials varying from −90 to +180 mV, by 10-mV step for each heterotetramers. The external and internal solutions contained 12 and 120 mM K+, respectively. (B) Normalized G-V curves for each heterotetramer. The conductance was calculated using the peak tail currents measured in symmetrical 120 mM K+ for the concatemers 4wt (black squares, n = 4), 1ILT/3wt (red circles, n = 7), averaged of dimers wt/wtILT and wtILT/wt (blue up triangles, n = 9 total), 3ILT/1wt (green down triangles, n = 5), 4ILT (yellow diamonds, n = 5), and Shaker zH4 Δ6-46 ILT (open diamonds, n = 5), respectively. The inset shows the V1/2 of the G-V curves plotted against the number of ILT subunits. The straight line represents a linear fit to the data. The values for 4wt were taken from Fig. 4 of Gagnon and Bezanilla (2009).
	Figure 2. Effect of the stoichiometry of ILT subunits on Shaker activation and deactivation kinetics. (A) Activation time constants (τact) and (B) deactivation time constants (τdeact) of the heterotetramers compared with that of 4ILT. The weighted τact as a function of the activating membrane potential is shown. The inset illustrates a current trace (black) at +160 mV from an oocyte expressing 4ILT, a fit with a single (red) and double (blue) exponential function. The vertical dotted line indicates the time position for the beginning of the fit. The internal and external solutions contained 120 and 12 mM K+, respectively. The weighted τdeact from an activating pulse at +160 mV plotted against the returning membrane potential. The internal and external solutions contained 120 and 50 mM K+, respectively. 4wt (black squares, n = 5), 1ILT/3wt (red circles, n = 3), 2ILT/2wt (blue up triangles, n = 3), 3ILT/1wt (green down triangles, n = 3), 4ILT (yellow diamonds, n = 4). Data from 4wt was taken from Fig. 4 of Gagnon and Bezanilla (2009).
	Figure 3. Single channel conductance is not modified by the stoichiometry of ILT subunits. (A) Representative traces of single channel activity measured in an oocyte expressing Shaker ILT at +50 mV (left) and −90 mV (right). The closed and open channel current levels are indicated as c and o, respectively. (B) All point histograms of single channel group of selected traces at +50 mV (left) and −90 mV (right). (C) Single channel current amplitude versus voltage relationship for the heterotetramers and Shaker ILT. Shaker ILT (open diamonds, n = 4), 3ILT/1wt (green down triangles, n = 3), 2ILT/2wt (blue up triangles, n = 3), and 1ILT/3wt (red circles, n = 3). The internal and external (pipette) solutions both contained 120 mM K+. Patch-clamp experiment.
	Figure 4. Temperature and voltage dependence of the activation and deactivation kinetics of 4ILT. (A) Voltage dependence of τact at four temperatures, as indicated, for a typical oocyte expressing 4ILT. The top and bottom panels represent the fast and slow component of the activation, respectively. The error bars are the error on the fit. The top panel inset shows family of ionic current traces measured at 19 (top) and 5°C (bottom), for a typical oocyte expressing 4ILT (oocyte 3, 11/03/09). The bottom panel inset shows the proportion of the fast component (oocyte 2, 05/02/09). Averaged voltage dependence of the weighted τact (B) and τdeact (D) at four temperatures for 4ILT. Data from all the oocytes combined, each oocyte was tested for at least two temperatures. For τact, 19°C (n = 8), 15°C (n = 4), 10°C (n = 4), and 5°C (n = 3) and for τdeact, 19°C (n = 6), 15°C (n = 5), 10°C (n = 4), and 5°C (n = 4). In both cases, the internal solutions contained 120 mM K+; the external solutions contained 12 and 50 mM K+ for activation and deactivation, respectively. The error bars are the SEM. Arrhenius plot of the ln of rate of activation (ln(1/τact)) (C) at +180 mV and that of the rate of deactivation (ln(1/τdeact)) (E) at 0 mV for 4ILT. The slow (squares), fast (circles), and weighted (triangles) components are shown. For activation, the slopes and intercepts for a linear fit to the data were −9800 ± 1800 (s−1/K−1) and 38 ± 6 (s−1), −13000 ± 800 (s−1/K−1) and 50 ± 3 (s−1), and −12900 ± 2200 (s−1/K−1) and 49 ± 8 (s−1) for the three components, respectively. For deactivation, the slopes and intercepts for a linear fit to the data were −8500 ± 500 (s−1/K−1) and 35 ± 2 (s−1), −5700 ± 1600 (s−1/K−1) and 26 ± 6 (s−1), and −2200 ± 100 (s−1/K−1) and 12.6 ± 0.5 (s−1) for the three components, respectively. The activation energy (Eaf) for the forward transition was calculated from the slope (−Ea/R) at 19.5, 25.8, and 25.6 kCal/mol and that of the backward transition (Eab) at 16.9, 11.3, and 4.4 kCal/mol.
	Figure 5. Determination of the chemical component of the free energy and effect of the temperature on the conductance in 4ILT. (A) Global fitting of the voltage dependence of weighted 1/τact and 1/τdeact for 4ILT. The data were fitted globally with the ratio of the forward and backward components of Eq. 1, α for activation and β for deactivation (see Materials and methods for full equations), assuming that α0 = β0. zα was found to be null. The lines represent the curve from the best fit. The results of zβ, ΔG for 4ILT, and the heterotetramers are listed on Table II. ΔH was calculated from Ea and z (ΔH = (Eaf − Eab) + (zα − zβ)FV) for V = 0, and TΔS was calculated from ΔG and ΔH (see Table II). (B) Right shift of the G-V with a decrease in temperature in 4ILT. Normalized G-V curve for a typical oocyte measured at temperatures of 19, 15, 10, and 5°C. The shift from 19 to 5°C was in averaged 7 ± 3 mV (n = 4). The conductance was calculated using the peak tail currents. The external and internal solutions contained 12 and 120 mM K+, respectively.
	Figure 6. Arrhenius plots of the forward and backward transition for the ILT heterotetramers. The ln(1/τact) (A) and ln(1/τdeact) (B) are plotted against 1/T (K−1). The slopes of the curve (−Ea/R), in which R is the gas constant (1.985 Cal·mol−1·K−1), are used to calculate Ea for each heterotetramer (see Table I). 1ILT/3wt (red circles), 2ILT/2wt (blue up triangles), 3ILT/1wt (green down triangles), 4ILT (yellow diamonds), and 1ILT/3neu (black squares). Data for activation were taken at +70, +60, +150, +180, and +100 mV for 1ILT/3wt, 2ILT/2wt, 3ILT/1wt, 4ILT, and 1ILT/3neu, respectively, and at +60, +10, 0, 0, and +30 mV for deactivation for 1ILT/3wt, 2ILT/2wt, 3ILT/1wt, 4ILT, and 1ILT/3neu, respectively.
	Figure 7. Conductance and kinetics of the single voltage sensor Shaker ILT. (A) Normalized G-V curve for the single voltage sensor Shaker ILT. Data from 4wt (squares), 1wt/3neu (stars), 1ILT/3wt (circles, n = 7), and 1ILT/3neu (diamonds, n = 4) are shown. The ones from 4wt and 1wt/3neu were taken from Gagnon and Bezanilla (2009). The internal and external solutions contained both 120 mM K+, respectively. (B) Kinetics of activation (τact, open symbols) and deactivation (τdeact, filled symbols) of 1ILT/3neu measured at room temperature. The slow (squares), fast (circles), and weighted (top triangles) components are shown. In both cases, the internal solutions contained 120 mM K+; the external solutions contained 12 and 50 mM K+ for activation and deactivation, respectively.
	Figure 8. Temperature and HP effects on 4ILT. (A) The voltage dependence of the charge movement in Shaker ILT, 4ILT compared with that of Shaker. Gating currents were measured in oocytes expressing Shaker, Shaker ILT, and 4ILT, integrated over time to get the transferred charge at different Vm and obtain the Q-V curve. HP was −90 mV, subtraction was made online with the P/−4 protocol, and subtracting HP was −120 mV. Each oocyte’s Q-Vs were individually fitted with a Boltzmann relationship (Q = Qmin + Qmax/(1 + exp[−z(V1/2 − Vm)F/RT]) in which z, F, R, and T have their usual meanings. The curves were normalized and baselined so that the charge at hyperpolarizing Vm is null. The V1/2 have values of −50 ± 2, −77 ± 2, and −77 ±1 mV and z of 2.6 ± 0.3, 2.3 ± 0.5, and 2.4 ± 0.4 for Shaker (filled circles, n = 4), Shaker ILT (filled squares, n = 4), and 4ILT (open squares, n = 4), respectively. The external and internal solutions for gating current measurement were used and both contained 115 mM NMG+. (B and C) No effect of temperature on the steady-state charge movement in 4ILT. Q-V curves (QON and QOFF) at various temperatures, as indicated, for a typical oocyte expressing 4ILT (B). Filled symbols correspond to the QON and the open symbols to the QOFF; squares (19°C), circles (15°C), up triangles (10°C), and down triangles (5°C). Averaged Q/Qmax-V curves (n = 4) for 4ILT (C). The averaged V1/2 were −76 ± 1 and −75 ± 1 (QON and QOFF, 19°C, squares), −75 ± 1 and −78 ± 1 (QON and QOFF, 15°C, circles), −76 ± 1 and −77 ± 1 (QON and QOFF, 10°C, up triangles), −75 ± 1 and −80 ± 1 mV (QON and QOFF, 5°C, down triangles), respectively. The slope factors were all comprised between 2.1 and 3.1 ± 0.1–0.3. The external and internal solutions contained 12 and 120 mM K+, respectively. (D) Temperature decrease slows the kinetics of ON gating charge in 4ILT. Traces from −140 mV to +30, 0, −30, −60, and −90 mV are shown, as indicated, at 19 and 10°C (top). The rates (1/α) of ON gating currents measured at different temperatures are plotted against Vm (bottom). The lines represent the global fit of the data, with a z of 0.4 and ΔG of 2 kCal/mol. (E) No effect of the HP on the charge movement or conductance in 4ILT. Q/Qmax-V (squares) curves for a typical oocyte expressing 4ILT when held at two HP. G/Gmax-V (circles) curves measured for 4ILT (n = 6) when measured at two HP. When HP was −90 mV, subtraction was made with the P/−4 protocol, and subtracting HP was −120 mV. When HP was 0 mV, subtraction was made using the P/−8 protocol, and the subtracting HP was +10 mV (no ionic current are observed at this Vm). The solutions contained both 115 mM NMG+ for charge movement measurement but under 12 and 120 mM K+ in the external and internal solutions, respectively, for ionic current measurements.
	Figure 9. Predictions of the model for ionic currents in symmetrical 120 mM K+ at room temperature (19–20°C) for 4ILT and 1ILT/3neu compared with experimental data. Experimental data are shown on the left, and the right panel illustrates the predictions of the kinetic model illustrated in Scheme 1. The HP was −90 mV, and steps were elicited to the annotated Vm. The rate constants and equivalent valence in electronic charge units are shown in Table III.

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