Figure 5.
Effects of manipulating cholesterol on the equilibrium and kinetic properties of HCN channel gating in nociceptor DRG neurons. (A) Application of WSC decreased the current amplitude of HCN currents in the same-patch experiment. Double-exponential fittings (in red) of the current traces at −140 mV are highlighted. (B) Amplitude of HCN currents elicited by a saturating hyperpolarizing voltage was used for the graph comparing the before and after treatments. Data shown are mean + SEM, n = 10 patches, *P = 0.01, two-sided paired t test. (C) Effects of the acute mβCD treatment on current amplitude of HCN currents, recorded from the same patch, showing opposite directional changes as in panel B. Data shown are mean ± SEM, n = 5 patches, **P = 0.005, two-sided paired t test. (D) Representative G-V relationship of HCN channel activation recorded from same-patch experiments before and after 5 min of 0.5 mg/ml WSC application, in the presence or absence of 0.5 mM additional cAMP. (E and F) Summary of the parameters derived from Boltzmann fitting of the G-V relationship of HCN channel activation before and after the WSC treatment: V1/2 in panel E (P = 2e−5 without cAMP, P = 0.01 with cAMP) and slope factors (Vs) in panel F; n.s., no statistical significance. n = 7 cells without added cAMP and n = 5 with added 0.5 mM cAMP, two-sided paired t test. (G and I) HCN channel currents elicited by different hyperpolarizing voltages were shown with double-exponential fits to highlight the acceleration of channel activation by mβCD (G) and the slowdown of channel activation by WSC (I). (H and J) Summary graph illustrating the effects of mβCD (H, n = 5, P = 3e−3, 6e−5, 4e−5, 1e−3, and 1e−4 for 100 mV, −110 mV, −120 mV, −130 mV, and −140 mV) and WSC (J, n = 8, P = 0.02, 0.02, 0.016, 0.18, and 1e−3 for 100 mV, −110 mV, −120 mV, −130 mV, and −140 mV) on the τ1 of channel activation at different voltages. Data shown are mean ± SEM, *P < 0.05, **P < 0.01, two-sided paired t test. Refer to the image caption for details. The image consists of two panels labeled A and B. Panel A shows current traces of H C N channels before and after treatment with 0.5 millimolar cyclic adenosine monophosphate (c A M P) and methyl-beta-cyclodextrin (m beta C D). The traces are color-coded: black for before treatment and blue for after treatment. The time constant (Tau 1) decreases from 105 milliseconds to 57 milliseconds after m beta C D treatment. Panel B presents a graph comparing the activation time constant (Tau activation) of H C N channels at different voltages before and after treatment. The horizontal axis represents voltage in millivolts (m V), ranging from minus100 to minus 130 m V, and the vertical axis represents the activation time constant in milliseconds (m s). The graph shows two data series: one in red for before treatment and one in blue for after treatment, indicating a decrease in activation time constant after treatment. All data are approximate.

Effects of manipulating cholesterol on the equilibrium and kinetic properties of HCN channel gating in nociceptor DRG neurons. (A) Application of WSC decreased the current amplitude of HCN currents in the same-patch experiment. Double-exponential fittings (in red) of the current traces at −140 mV are highlighted. (B) Amplitude of HCN currents elicited by a saturating hyperpolarizing voltage was used for the graph comparing the before and after treatments. Data shown are mean + SEM, n = 10 patches, *P = 0.01, two-sided paired t test. (C) Effects of the acute mβCD treatment on current amplitude of HCN currents, recorded from the same patch, showing opposite directional changes as in panel B. Data shown are mean ± SEM, n = 5 patches, **P = 0.005, two-sided paired t test. (D) Representative G-V relationship of HCN channel activation recorded from same-patch experiments before and after 5 min of 0.5 mg/ml WSC application, in the presence or absence of 0.5 mM additional cAMP. (E and F) Summary of the parameters derived from Boltzmann fitting of the G-V relationship of HCN channel activation before and after the WSC treatment: V1/2 in panel E (P = 2e−5 without cAMP, P = 0.01 with cAMP) and slope factors (Vs) in panel F; n.s., no statistical significance. n = 7 cells without added cAMP and n = 5 with added 0.5 mM cAMP, two-sided paired t test. (G and I) HCN channel currents elicited by different hyperpolarizing voltages were shown with double-exponential fits to highlight the acceleration of channel activation by mβCD (G) and the slowdown of channel activation by WSC (I). (H and J) Summary graph illustrating the effects of mβCD (H, n = 5, P = 3e−3, 6e−5, 4e−5, 1e−3, and 1e−4 for 100 mV, −110 mV, −120 mV, −130 mV, and −140 mV) and WSC (J, n = 8, P = 0.02, 0.02, 0.016, 0.18, and 1e−3 for 100 mV, −110 mV, −120 mV, −130 mV, and −140 mV) on the τ1 of channel activation at different voltages. Data shown are mean ± SEM, *P < 0.05, **P < 0.01, two-sided paired t test.

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