Optical control of an ion channel gate

Abstract

The powerful optogenetic pharmacology method allows the optical control of neuronal activity by photoswitchable ligands tethered to channels and receptors. However, this approach is technically demanding, as it requires the design of pharmacologically active ligands. The development of versatile technologies therefore represents a challenging issue. Here, we present optogating, a method in which the gating machinery of an ATP-activated P2X channel was reprogrammed to respond to light. We found that channels covalently modified by azobenzene-containing reagents at the transmembrane segments could be reversibly turned on and off by light, without the need of ATP, thus revealing an agonist-independent, light-induced gating mechanism. We demonstrate photocontrol of neuronal activity by a light-gated, ATP-insensitive P2X receptor, providing an original tool devoid of endogenous sensitivity to delineate P2X signaling in normal and pathological states. These findings open new avenues to specifically activate other ion channels independently of their natural stimulus.

Keywords: azobenzene photoswitch; purinergic receptors.

Fig. 1.

Tethered MEA-TMA turns on and off the P2X2 channel by light. (A) Chemical structure and cis-trans isomerization of MEA-TMA. (B) Whole-cell currents evoked by light at the indicated wavelengths in HEK cells expressing the indicated constructs that were prior-treated to MEA-TMA in the dark. For V48C, labeling was performed in the presence of ATP. (C) Screening for all constructs showing current density recorded in the dark, followed by 365- (violet bars) and 525-nm (green bars) light illuminations (n = 4–7 cells; mean ± SEM). (D) Mapping of switchable positions viewed from the extracellular side along the threefold axis of symmetry of a closed-channel state P2X2 homology model reveals trans- and cis-openers, colored as green and violet sticks, respectively. Gray- and black-colored sticks indicate, respectively, selected and labeled residues with no effect upon light irradiation. TM1 and TM2 α-helices from the same subunit are also indicated.

Fig. 2.

Kinetics and bistability of azobenzene isomerization attached at the I328C mutant. (A) Increasing 525-nm light intensity (green bar) to a cell expressing the I328C mutant tethered to MEA-TMA increases activation rates of whole-cell current. Data were fitted by single exponential decay functions (in red). (B) Bar plot summarizing data presented in A shows substantial change of time constant (τ) but not of light-induced current amplitude (normalized to 0.36 mW/mm²) (n = 4 cells; error bars are SEM). (C) Bistability of light-induced currents revealed in the dark following brief photoactivation at 525 nm (green bars, light intensity was set to 4.5 mW/mm²) and brief inactivation at 365 nm (violet bars) in a cell expressing the I328C mutant tethered to MEA-TMA. Note that the basal current is stable in the dark until a new photoactivation of similar amplitude occurs.

Fig. 3.

Tethered MEA-TMA photomodulates ATP response of P2X2 channels. (A) Whole-cell currents evoked by ATP (EC₅₀) modulated by light in the same cells as those shown in Fig 1B. For I328C and I332C, cells were prior-irradiated with a short 4-s lasting pulse of 365-nm light to induce channel closure. (B) Same view as in Fig. 1D, in which trans- and cis-blockers colored, respectively, as green and violet sticks, are mapped on a P2X2 model in an ATP-bound, open-channel state. Gray- and black-colored sticks indicate, respectively, selected and labeled residues with no effect upon light irradiation on ATP response. (C) ATP dose–response curves recorded at 365-nm (violet) and at 525-nm light (green) from cells expressing the I328C mutant tethered with MEA-TMA (n = 5 cells; mean ± SEM). Currents were normalized to 30 μM ATP at 365 nm. (Inset) Typical superimposed ATP traces at the indicated illumination wavelengths.

Fig. 4.

Critical role of the photoswitch-positive charge for labeling P2X2 channels. (A) Chemical structures of MEA-TMA analogs. (B) Whole-cell currents evoked by light in cells expressing the I328C mutant tethered with MAQ, MEA-SO₃, or MEA-OMe, as indicated. (C) Bar plot showing photocurrent amplitudes elicited by 525-nm light normalized to currents evoked by a saturating concentration of ATP (recorded at 365-nm light) in cells expressing the I328C mutant tethered to the indicated MEA-TMA analogs. Labeling was performed with either 200 μM (white bars) or 80 μM (gray bars) of the analogs (n = 3–7 cells; mean ± SEM).

Fig. 5.

An ATP-insensitive light-gated cation channel drives synaptic activity and action potential firing in neurons. (A) The K69A/I328C mutant tethered to MEA-TMA responds to light (Left), but not to ATP (Right, 100 μM) in HEK cells. (B) Current-voltage curves for the same construct recorded in different extracellular solutions in HEK cells (Man, mannitol; Na-Ise, sodium isethionate; Ca, calcium). Light-gated currents were obtained after subtracting photocurrents recorded at 525-nm light (intensity was set to 4.5 mW/mm²) to those obtained at 365-nm light (for NMDG, current shown was recorded 60 s after light switching). (C) Whole-cell current from two example recordings (with moderate and robust synaptic activities) evoked by light in hippocampal neurons expressing the K69/I328C mutant following brief application of 100 μM MEA-TMA (solid lines). (D) Bar plot summarizing the recorded current densities (mean ± SEM for the indicated number of experiments). (E) Extracellular recording from hippocampal neurons expressing the K69/I328C mutant showing action-potential (AP) firing triggered by 525-nm light illumination. Cells were labeled with MEA-TMA (30 μM) under 365-nm light to maintain channels in nonconducting states. (F) Bar plot summarizing AP rates, defined as the number of AP per second, recorded under 365-nm light (violet bar) and 525-nm light (green bar) in experiments similar to that shown in E (n = 4, mean ± SEM). Rates, calculated every 5 s, were collected just before 525-nm excitation, and at the peak, within the first minute of the 525-nm excitation. Measurement of statistical significance is based on paired Student t tests; *P < 0.05.