![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.](https://cdn.rupress.org/rup/content_public/journal/jgp/136/5/10.1085_jgp.201010487/4/jgp_201010487_rgb_fig8.jpeg?Expires=1767727701&Signature=BeFJlqxQcu5IzY5-h7MQ5EcwJWtBn0m-1VYXw3dglErTfdk5Gog3aXnq-mK1sGP~P6shzu55dKNdy8tMZIJ9SipXpqC2oCEhAXNJMpY1lBvGiT0YbXIxzaUuy-9irGEz0R4KomwCz8hNcht83Se7dcQnjMrVIVE37VfHa7zGNRKxoL-ml7i4I6HEAzCCT2eAFc7joZCgEi9gJHWXndbH0aPY8qeROW3DEbG7hdnGNH1NdHDUF03tfY04~d-wCv40T4tZNzKvchTOFgqiNxJhIeenuk3ocBum5Ztj-JoA7-xsqGMDIfbVMwSl7CcKsWutvZHH6XdpmZNu5eU0DpLwJQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
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.
