Optical control of trimeric P2X receptors and acid-sensing ion channels

Abstract

P2X receptors are trimeric membrane proteins that function as ion channels gated by extracellular ATP. We have engineered a P2X2 receptor that opens within milliseconds by irradiation at 440 nm, and rapidly closes at 360 nm. This requires bridging receptor subunits via covalent attachment of 4,4'-bis(maleimido)azobenzene to a cysteine residue (P329C) introduced into each second transmembrane domain. The cis-trans isomerization of the azobenzene pushes apart the outer ends of the transmembrane helices and opens the channel in a light-dependent manner. Light-activated channels exhibited similar unitary currents, rectification, calcium permeability, and dye uptake as P2X2 receptors activated by ATP. P2X3 receptors with an equivalent mutation (P320C) were also light sensitive after chemical modification. They showed typical rapid desensitization, and they could coassemble with native P2X2 subunits in pheochromocytoma cells to form light-activated heteromeric P2X2/3 receptors. A similar approach was used to open and close human acid-sensing ion channels (ASICs), which are also trimers but are unrelated in sequence to P2X receptors. The experiments indicate that the opening of the permeation pathway requires similar and substantial movements of the transmembrane helices in both P2X receptors and ASICs, and the method will allow precise optical control of P2X receptors or ASICs in intact tissues.

Fig. 1.

Light activation of P2X2 receptors. (A) Models of rat P2X2 receptor showing closed (Left) and open (Right) conformations. (Upper) Space-fill of trimeric holoprotein. (Lower) Ribbon representation of TM domains, from extracellular side. The positions of P329 are indicated in green. (B) BMA, in its cis state (Left) and trans state (Right). (C) Aligned sequences of second transmembrane domains of rat P2X2 and P2X3 receptors, and human ASIC1a. (D) Light-activated P2X2[P329C] receptors. Currents were evoked by illumination at 440 nm (2 s, blue bar) and turned off by illumination at 360 nm (2 s, violet bar). Light-induced currents were 35% ± 4% (n = 11) of the amplitude of maximum currents evoked by ATP. There was no effect of 440-nm or 360-nm illumination at wild-type P2X2 receptors or at P2X2[P329S] receptors (middle traces), but normal responses to ATP. When the P329C mutation was combined with K69A mutation, ATP (100 μM, 2 s) had no effect (right trace), whereas light-induced currents were present. Preincubated for 10–12 min with BMA (10 μM), in each case. ATP was 3, 10, 10, and 100 μM (left to right). Currents normalized to the peak amplitude evoked by ATP, except that P329C/K69A uses the same scale as P329C. Actual peak amplitudes were as follows: P329C 1879 pA, wild type 1937 pA, P329S 1954 pA, and P329C/K69A 1360 pA.

Fig. 2.

Same permeation properties of light-gated and ATP-gated P2X2 receptors. (A) Single-channel currents from cells expressing wild-type or P329C receptors. (Upper) Wild-type receptors in ATP (3 μM). (Lower) P2X2[P329C] receptors after treatment with BMA and exposure to 440 nm light. (B) All-points histograms for wild-type receptors activated by ATP, P329C receptors activated by ATP, P329C receptors after BMA treatment activated by light, and P329C receptors after BMA treatment activated by ATP. (C) Current–voltage plots for currents evoked by light (blue) and ATP (black) from cell expressing the P2X2[P329C] receptor. (D) Current–voltage plots from two cells expressing the P2X2[P329C] receptor activated by light (blue traces) or ATP (black and gray traces). Shift in reversal potential observed between external calcium (112 mM) and sodium (154 mM) was similar for P2X2[P329C] receptors gated by light (blue) and by ATP (black).

Fig. 3.

Optical control of heteromeric P2X2/3 receptors. (A, Left) Whole-cell recordings from HEK293 cells expressing wild-type P2X3 subunit cDNA show rapidly desensitizing currents elicited by αβmeATP (30 μM) but no effect of light (440 nm 2 s, 360 nm 2 s). (A, Right) Cells expressing mutant P2X3[P320C] subunit cDNA showed rapidly desensitizing currents evoked by 440 nm light (8% ± 3% (n = 5) of amplitude of currents evoked by αβmeATP). (B, Left) Recordings from cells cotransfected with wild-type P2X2 and P2X3 subunit cDNAs show slowly desensitizing currents evoked by αβmeATP (30 μM) but no effect of light. (B, Right) Light-evoked currents in cell coexpressing wild-type P2X2 subunit cDNA and P2X3[P320C] subunit cDNA. Heteromeric P2X2/3 receptor current was 16% ± 4% (n = 8) the amplitude of currents evoked by αβmeATP (each measured 2 s after beginning of application). Traces in A and B normalized to the peak current amplitude induced by αβmeATP (amplitudes were as follows: wild-type P2X3, 1,845 pA; P2X3[P320C], 2,394 pA; P2X2 wild type/P2X3 wild type, 860 pA; P2X2 wild type/P2X3[P320C], 917 pA) or by light (amplitudes were as follows: P2X3[P320C], 500 pA; P2X2 wild type/P2X3[P320C], 193 pA).

Fig. 4.

Light activates heteromeric P2X2/3 receptors in PC12 cells. (A) PC12 cells transfected with P2X3[P320C] depolarized by 440 nm irradiation and switched off by 360 nm light. These cells respond to αβmeATP with a sustained depolarization, indicative of heteromeric P2X2/3 receptors. (B) Cells transfected with wild-type P2X3 receptors do not respond to light but are depolarized by αβmeATP. (C) Native PC12 cells (mock transfected with GFP) show responses to ATP (P2X2 receptors) but no effect of light or αβmeATP. ATP and αβmeATP concentrations 30 μM.

Fig. 5.

Optical control of trimeric ASIC1 channels. (A) Pore of trimeric chick ASIC1 channels, depicted in desensitized (Left, Proteing Data Bank ID 3IJ4) and open (Right, Protein Data Bank ID 4FZ1) states, showing transmembrane domains viewed from the outside. The residue corresponding to G430 is shown in green. (B) CHO cells expressing wild-type human ASIC1a subunit cDNAs show large inward currents in pH 5.3 but no effect of light (preincubated with BMA). (C) G430C and I428C ASIC1b receptors showed light-activated currents. For G340C, light induced currents were 15% ± 9% (n = 5) the amplitude of currents evoked by pH 5.3, and for I428C this was 10% ± 1% (n = 4). Traces to peak current amplitude induced by pH 5.3 (amplitudes were as follows: wild type, 3,866 pA; G430C, 1,986 pA; I428C, 3,148 pA) or by light (amplitudes were as follows: G430C, 969 pA; I428C, 448 pA).