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J Gen Physiol
2014 May 01;1435:633-44. doi: 10.1085/jgp.201311112.
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State-dependent and site-directed photodynamic transformation of HCN2 channel by singlet oxygen.
Gao W
,
Su Z
,
Liu Q
,
Zhou L
.
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Singlet oxygen ((1)O2), which is generated through metabolic reactions and oxidizes numerous biological molecules, has been a useful tool in basic research and clinical practice. However, its role as a signaling factor, as well as a mechanistic understanding of the oxidation process, remains poorly understood. Here, we show that hyperpolarization-activated, cAMP-gated (HCN) channels--which conduct the hyperpolarization-activated current (Ih) and the voltage-insensitive instantaneous current (Iinst), and contribute to diverse physiological functions including learning and memory, cardiac pacemaking, and the sensation of pain--are subject to modification by (1)O2. To increase the site specificity of (1)O2 generation, we used fluorescein-conjugated cAMP, which specifically binds to HCN channels, or a chimeric channel in which an in-frame (1)O2 generator (SOG) protein was fused to the HCN C terminus. Millisecond laser pulses reduced Ih current amplitude, slowed channel deactivation, and enhanced Iinst current. The modification of HCN channel function is a photodynamic process that involves (1)O2, as supported by the dependence on dissolved oxygen in solutions, the inhibitory effect by a (1)O2 scavenger, and the results with the HCN2-SOG fusion protein. Intriguingly, (1)O2 modification of the HCN2 channel is state dependent: laser pulses applied to open channels mainly slow down deactivation and increase Iinst, whereas for the closed channels, (1)O2 modification mainly reduced Ih amplitude. We identified a histidine residue (H434 in S6) near the activation gate in the pore critical for (1)O2 modulation of HCN function. Alanine replacement of H434 abolished the delay in channel deactivation and the generation of Iinst induced by photodynamic modification. Our study provides new insights into the instantaneous current conducted by HCN channels, showing that modifications to the region close to the intracellular gate underlie the expression of Iinst, and establishes a well-defined model for studying (1)O2 modifications at the molecular level.
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24733837
???displayArticle.pmcLink???PMC4003188 ???displayArticle.link???J Gen Physiol ???displayArticle.grants???[+]
Figure 1. Effects on Ih current by photodynamic transformation. (A) Bright field (left) and fluorescence (right) images of a membrane patch expressing mHCN2 channels. Bottom pictures show a membrane patch from an uninjected cell. 0.5 µM FITC-cAMP was applied to the bath. (B; top) The voltage protocol for channel activation and the timing of 100-msec laser pulse. (Bottom) Representative current traces before (no. 3 in C) and after the application of laser pulses (nos. 6 and 9 in C). (C) Normalized Ih amplitude versus stimulation index. Voltage step was delivered every 15 s. (D) Normalized time constant of deactivation. *, P < 0.05 in C and D. (E; left) The voltage protocol for collecting I-V curve. (Middle) Current traces before the laser treatment. (Right) Current traces after the laser treatment. (F) I-V curves without laser treatment. The V1/2 values are −126.7 ± 3.2 mV (n = 8; control without cAMP), −109.4 ± 2.6 mV (first I-V in 0.5 µM FITC-cAMP), and −112.5 ± 2.4 mV (second I-V in 0.5 µM FITC-cAMP). The averaged shift in V1/2 is −3.1 ± 1.7 mV (n = 7) and not significant (one-way repeated measures ANOVA). (G) I-V curve with laser treatment. The V1/2 values are −114.6 ± 1.6 mV (I-V in 0.5 µM FITC-cAMP before laser) and −120.4 ± 1.2 mV (I-V in 0.5 µM FITC-cAMP after laser treatments). The averaged shift in V1/2 is −5.8 ± 0.8 mV (n = 6) and significant.
Figure 2. Photodynamic transformation slows down channel deactivation and enhances voltage-insensitive Iinst. (A) Current traces before (black; no. 3 in B) and after (blue; no. 7 in B) laser treatments. Arrow indicates Iinst. Gray arrows point out close-up view over the current traces during channel deactivation. (B) Percentage of Iinst (top) or time constant of channel deactivation (bottom) versus episode number. Laser pulses (filled blue triangle) were given every 15 s after the third episode. FITC-cAMP, n = 19; cAMP, n = 7. *, P < 0.05.
Figure 3. Photochemically generated Iinst has key biophysical features of canonical HCN channel. (A) Voltage protocol (top) and current traces for measuring pK+/pNa+ of Ih current. (B) I-V curves of Ih current. (C) Voltage protocols (top) and current traces of Iinst. (D) I-V curves of Iinst. (E) ZD7288 and Cs+ interact with different regions in the HCN pore. (F) Iinst was generated by applying laser pulses (nos. 3–5). 60 µM ZD7288 was added to the bath solution after number 9. (G) 2 mM Cs+ was added to the pipette solution. A voltage step to +60 mV released the Cs+ block. Current traces: black, control; blue, after three laser pulses. Current traces in A–C were measured in the absence of ligands (cAMP or FITC-cAMP). 0.5 µM FITC-cAMP was present in F and G.
Figure 4. Photochemical transformation of the HCN channel is oxygen dependent. Solutions used in the experiments were degassed and then purged with pure N2 or O2. (A and B) Current traces before (black; no. 3 in C and D) and after laser pulses (blue; no. 8 in C and D). *, the group of O2 is significantly different from the data groups of Normal air + trolox and N2 (one-way ANOVA). (C and D) τdeactivation or percentage of Iinst versus episode number. 0.5 µM FITC-cAMP was applied to the bath solution. *, the groups of O2 and normal air are significantly different from the other two data groups.
Figure 5. 1O2 mediates photochemical transformation of the mHCN2 channel. (A) Construction of the HCN2-SOG fusion channel. (B; top) Bright field and fluorescence images of a membrane patch expressing HCN2-SOG channels. (Bottom) Excitation and emission spectra of purified mini-SOG protein. (C) Timing of the laser pulse and the voltage protocol. (D) Current traces before (black; no. 3 in E) and after (blue; no. 7 in E) laser treatments. (E) τdeactivation or percentage of Iinst versus episode number. Neither cAMP nor FITC-cAMP was applied. *, P < 0.05.
Figure 6. 1O2 modification of the mHCN2 channel is state dependent. (A) Schematic drawings of closed and open channels. (B) Laser pulses were delivered preceding the hyperpolarization step. (C) Current traces before (black; no. 3 in D and E) and after (blue; no. 7 in D and E) laser treatments. (D and E) Normalized τdeactivation or percentage of Iinst versus episode number. 0.5 µM FITC-cAMP was applied to the bath. *, P < 0.05.
Figure 7. H434 is critical for 1O2 modification of the mHCN2 channel. (A) Structure model of the mHCN2 pore (based on Kv1.2-2.1 chimeric structure) (Long et al., 2007). (B) Current traces of mHCN2/H434A mutant channel in the presence of 0.5 µM FITC-cAMP. A series of hyperpolarizing voltage steps in a −10-mV interval was used to activate Ih. (C) I-V curves of mHCN2/H434A before (black) and after (blue) laser treatment. Current amplitudes were normalized to the maximal level before laser treatment. V1/2 values (with laser treatment) are −121.4 ± 1.7 mV (before laser in FITC-cAMP) and −129.7 ± 1.3 mV (after laser). The corresponding ΔV1/2 is −8.3 ± 2.0 mV (n = 7) and significant (one-way repeated measures ANOVA). As a control, the V1/2 values in the presence of FITC-cAMP but without laser treatment are −119.6 ± 3.3 mV (first I-V) and −128.2 ± 4.1 mV (second I-V). The ΔV1/2 is −8.5 ± 1.6 mV (n = 8; significant). (D; top) Laser pulse. (Middle) Voltage protocol. (Bottom) Current traces before (black; no. 3 in D and E) and after (blue; no. 7 in D and E) laser treatments. (E) Normalized τdeactivation or percentage of Iinst versus episode number. WT, n = 19; mHCN2/H434A, n = 13. *, P < 0.05.
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