Idikuda V et al. (2018), Singlet oxygen modification abolishes voltage-d...

XB-ART-56404

J Gen Physiol 2018 Sep 03;1509:1273-1286. doi: 10.1085/jgp.201711961.

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Singlet oxygen modification abolishes voltage-dependent inactivation of the sea urchin spHCN channel.

Idikuda V , Gao W , Grant K , Su Z , Liu Q , Zhou L .

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Photochemically or metabolically generated singlet oxygen (1O2) reacts broadly with macromolecules in the cell. Because of its short lifetime and working distance, 1O2 holds potential as an effective and precise nanoscale tool for basic research and clinical practice. Here we investigate the modification of the spHCN channel that results from photochemically and chemically generated 1O2 The spHCN channel shows strong voltage-dependent inactivation in the absence of cAMP. In the presence of photosensitizers, short laser pulses transform the gating properties of spHCN by abolishing inactivation and increasing the macroscopic current amplitude. Alanine replacement of a histidine residue near the activation gate within the channel's pore abolishes key modification effects. Application of a variety of chemicals including 1O2 scavengers and 1O2 generators supports the involvement of 1O2 and excludes other reactive oxygen species. This study provides new understanding about the photodynamic modification of ion channels by 1O2 at the molecular level.

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Genes referenced: camp dtl

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	Figure 1. Photodynamic modification of the spHCN channel in complex with FITC-cAMP. (A) Top: Voltage protocol for channel activation (−100 mV) and tail current recording (+40 mV). Bottom: WT spHCN channels show strong voltage-dependent inactivation in the absence of cAMP (black). With cAMP, the spHCN channel opens without any obvious inactivation (blue). The definitions of Iinst, Ih, Imacro, Ih(tail), and Iss are illustrated. Background leak conductance was not subtracted. (B) Top: Bright-field image of a piece of membrane patch (indicated by an arrow) held within the glass recording pipette. Bottom: Fluorescence image of the spHCN channel in complex with 1 µM 8-FITC-cAMP. Membrane patches from uninjected oocytes (no channel expressed) only show fluorescence intensity at background level (not shown). (C) Top: Schematic drawing of the patch-clamp recording under the inside-out configuration. Bottom: Chemical structure of 8-FITC-cAMP. (D) Top: Voltage step protocol and the timing of laser pulse. Bottom: The last current trace before (black; trace 0) and after (with color; traces 1–8) laser pulse application. Numbers from 1 to 8 represent the order of applied laser pulses and the corresponding current traces. The prominent increases in Iinst after the third laser pulse and in Iss after the fourth laser pulse are labeled (magenta). (E) A zoomed view over the moment when laser pulses were applied. Red box in dashed lines represents the laser pulses with 100-ms duration. (F) Laser pulses have no obvious effects on the spHCN channel in complex with regular 1 µM cAMP. (G) Averaged results (n = 13) showing the effect of laser pulse on the amplitude of Imacro. The horizontal box (with laser pulse labeled) indicates the current episodes with laser pulse treatment. Current amplitudes were normalized to the maximal value measured in the presence of 10 µM cAMP before the application of laser pulses. (H) Averaged results (n = 13) showing the effect of laser pulses on the time constant of deactivation. ***, P ≤ 0.001. Error bars represent SEM.
	Figure 2. Ih and Iinst share similar sensitivity to ZD7288, an HCN channel–specific blocker, and K+/Na+ selectivity. (A) Top: Voltage protocol. Bottom: Four current traces in sequence showing control (no cAMP), 10 µM cAMP, the third trace with laser pulse (with 1 µM FITC-cAMP), after laser pulses stopped and washing off FITC-cAMP, and ZD7288 (100 µM). (B) Zoomed views showing the Iinst (top) and the Ih(tail) and Iss (bottom). (C) Averaged results showing that laser pulses applied in the presence of FTIC-cAMP increase the amplitude of Imacro, which can be blocked by ZD7288. (D) Averaged results showing that laser pulses applied in the presence of FTIC-cAMP increase the amplitude of Iinst, which can also be blocked by ZD7288. Error bars represent SEM. (E) Top: Voltage protocol for Ih activation (0 to −90 mV) and a voltage ramp (−50 to 50 mV) used for the measurement of reversal potential before light pulses. Bottom: Current traces recording from the same membrane patch with symmetrical [K+]in/[K+]out (magenta) or [Na+]in/[K+]out (blue). 10 µM cAMP was added to the bath solution on the intracellular side. (F) Cross-plot of current amplitude versus membrane potential during the voltage ramp for Ih. Averaged results: 1.3 ± 0.3 mV (K+i); −27.8 ± 1.2 mV (Na+i); n = 4. (G) Top: A voltage ramp (+40 to −20 mV) was used for the measurement of reversal potential of Iinst. Bottom: Current traces recorded from the same membrane patch with symmetrical [K+]in/[K+]out (magenta, left) or [Na+]in/[K+]out (blue, right). Current traces in light colors are recorded before laser pulses and mainly caused by nonspecific leak conductance. Current traces in dark colors are recorded after laser pulses stopped and in the absence of FITC-cAMP. (H) Cross-plot of current amplitude versus membrane potential during the voltage ramp for the Iinst after laser pulses. Averaged results: 0.9 ± 0.2 mV (K+i); −26.5 ± 0.9 mV (Na+i); n = 4.
	Figure 3. Photodynamic modification of the spHCN channel in the closed state and the sensitivity of modified spHCN channels to Cs+. (A) Top: Voltage command and the timing of laser pulses. Laser pulses were applied preceding the hyperpolarization voltage step when most of the channels should stay in the closed state. Bottom: Current traces recorded with 1 µM FITC-cAMP in the bath solution. The last control trace before laser pulse is shown in black and labeled 0. (B) Zoomed views of the region within the red box shown in A. (C) Averaged (n = 15) results showing the effect of laser pulses on the amplitude of macroscopic current. **, P ≤ 0.001. (D) Averaged (n = 15) results showing the effect of laser pulses on the time constant of deactivation. , P ≤ 0.05. Error bars represent SEM. (E) During the −100-mV voltage step, Cs+ applied to the extracellular side of the membrane blocks the spHCN current after photodynamic modification (laser pulse during voltage step). The following depolarizing voltage step from −100 to +40 mV released the block by Cs+ and revealed the effects of photodynamic modification. Photodynamic modification leads to slowdown in channel deactivation and increases in Ih(tail) and Iss. Black, last current trace before laser pulse. Green, traces with laser pulses. (F) Laser pulses were applied preceding the hyperpolarization voltage step. Cs+ blocks the macroscopic current at −100 mV. The voltage step from −100 to +40 mV released the Cs+ block and revealed the effects of photodynamic modification. Detailed analyses are shown in Fig. S5.
	Figure 4. spHCN/H462A mutant channel shows minimal responses to photodynamic modification. (A) Top: Voltage steps used for channel activation and tail current recording. Bottom: Current traces of the spHCN/H462A mutant channel recorded in the presence of 10 µM cAMP. (B) Averaged voltage-dependent activation curve of the WT (black, n = 5; V1/2, −63.2 ± 0.8 mV) and spHCN/H462A mutant (red, n = 6; V1/2, −62.2 ± 0.8 mV) channels. (C) The spHCN/H462A mutant channel shows minimal responses to laser pulses during the voltage step from 0 to −100 mV. A zoomed view of the current trace is shown below. (D) Laser pulses applied before the voltage step leads to small increases in macroscopic current but minimal effects on channel inactivation. A zoomed view of the current trace is shown below. (E) Averaged results showing the effect of laser pulses (applied during voltage step) on the Imacro amplitude of the WT (n = 13, black) and spHCN/H462A mutant (n = 5, red) channels. *, P ≤ 0.001. (F) Averaged results showing the effect of laser pulses (applied before voltage step) on the Imacro amplitude of the WT (n = 15, black) and spHCN/H462A mutant (n = 5, red) channels. *, P ≤ 0.001. Error bars represent SEM.
	Figure 5. Applications of a popularly used photosensitizer, Rose Bengal, and 1O2 quenchers (sodium azide and Trolox-C) support the involvement of 1O2. (A) Schematic drawing of the experiment configuration. Rose Bengal was applied to the bath solution and in contact with the intracellular side of the channel. (B) In the presence of 100 nM Rose Bengal, laser pulses were applied in the middle of the voltage step from 0 to −100 mV. The current traces recorded before the application of laser pulses are shown below. (C) In the presence of 100 nM Rose Bengal, laser pulses were applied preceding the voltage step to −100 mV. The current traces recorded before the application of laser pulses are shown below. (D) Averaged results showing the effect of laser pulses (during the voltage step) on the Imacro amplitude of the WT (n = 8, black) and spHCN/H462A mutant (n = 8, red) channels. (E) Averaged results showing the effect of laser pulses (before the voltage step) on the Imacro amplitude of the WT (n = 5, black) and spHCN/H462A mutant (n = 5, red) channels. (F) Averaged results showing the effect of sodium azide (n = 7, blue) and Trolox-C (n = 8, purple) on the photodynamic modification of WT spHCN channels. FITC-cAMP was the photosensitizer. (G) Averaged results showing the effect of Trolox-C (purple) on the photodynamic modification (n = 11) of WT spHCN mutant (n = 7, red) channels. Rose Bengal was the photosensitizer. For D–G, results are mean ± SEM; ***, P ≤ 0.001. Error bars represent SEM.
	Figure 6. The function of WT spHCN channel can be modulated by chemically generated 1O2 but not by hydroxyl or superoxide radicals. (A) Chemical generation of 1O2 by mixing hydrogen peroxide and sodium hypochlorite. (B) Top: Current traces of WT spHCN channel recorded under control condition (black), in the presence of 10 mM H2O2 only (red), or in the presence of a mixture of 10 mM H2O2 and 10 mM NaClO (green). Bottom: Current traces recorded before chemical applications. (C) The spHCN/H462A mutant channel shows no responses to the chemical mixture of H2O2 and NaClO. Bottom: Current traces recorded before chemical applications. (D) Averaged results showing the responses to H2O2 or H2O2+NaClO by WT (n = 3, black) and H462A mutant (n = 3, red) spHCN channels. Error bars represent SEM. (E) Reaction schemes of the chemical generation of hydroxyl or superoxide radicals. (F) The WT spHCN channel shows no response to the mixture of FeSO4 (1 mM) and H2O2 (15 mM; n = 5). Bottom: Current traces recorded before application of chemicals. (G) The WT spHCN channel shows no response to the mixture of xanthine (5 mM) and xanthine oxidase (15 mU; 6 min of incubation; n = 6). Bottom: Current traces recorded before chemical applications.

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