Photochemical control of endogenous ion channels and cellular excitability

Doris L Fortin¹, Matthew R Banghart², Timothy W Dunn¹, Katharine Borges¹, Daniel A Wagenaar³, Quentin Gaudry³, Movses H Karakossian⁴, Thomas S Otis⁴, William B Kristan³, Dirk Trauner² & Richard H Kramer¹

¹ Department of Molecular and Cell Biology, University of California Berkeley, 121 Life Sciences Addition, Berkeley, California 94720, USA.

² Department of Chemistry, University of California Berkeley, Latimer Hall, Berkeley, California 94720, USA.

³ Department of Biology, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92037, USA.

⁴ Department of Neurobiology, University of California, Los Angeles, 63-314 CHS Box 951763, Los Angeles, California 90095, USA.

PALs are derivatives of the photoisomerizable molecule azobenzene (Fig. 1a). Connected to one end of azobenzene is a protein-binding ligand, in this case a quaternary ammonium group (QA), which binds to the pore of K⁺ channels and blocks ion conduction. On the other end is an electrophilic group that enables covalent attachment of the photoswitch to nucleophilic amino acid side chains in the channel. We designed PALs comprising QA, azobenzene and one of three different electrophilic groups: acrylamide (PAL abbreviated AAQ), chloroacetamide (CAQ) or epoxide (EAQ; Fig. 1b; see Supplementary Methods online for a description of the synthesis). Binding of the QA to the K⁺ channel pore increases the local concentration of the electrophile at the surface of the channel. This promotes covalent attachment if the channel has a nucleophilic side chain 20 Å from the QA binding site, matching the length of the PAL molecule. Hence, the covalent attachment of PALs to channels is promoted by ligand binding, as in classical affinity labeling²³. After the photoswitch is covalently tethered, the QA can reach the pore and block ion conduction only when the azobenzene is in its elongated trans form, but not in its bent cis form (Fig. 1c). Thus, channels can be unblocked by exposure to 360–400-nm light, which photoisomerizes the azobenzene from trans to cis. The reverse cis to trans conversion, which restores channel block, occurs slowly in the dark ( = 5 min) and is accelerated by exposure to long-wavelength light (450–560 nm)^{10, 24}.

Figure 1. The PAL approach for imparting light sensitivity onto native ion channels. (a) PAL molecules consist of a photoisomerizable azobenzene group flanked by QA and a covalent attachment group (R). Exposure to 380-nm light isomerizes the azobenzene to its shorter cis form, whereas exposure to 500-nm light favors the trans configuration. (b) Chemical structure of PAL molecules and MAQ. PAL molecules contain a promiscuous reactive group (orange); MAQ contains a maleimide (blue) designed to react with an engineered cysteine. (c,d) Schematic of the generation of photoswitch-regulated K+ channels. A PAL whose QA (burgundy) is bound to the channel pore covalently attaches via its promiscuous reactive group (orange) to an endogenous nucleophile (Nu) on a K+ channel (c). For the SPARK channel, MAQ attaches via its maleimide (blue) to a genetically introduced cysteine in a Shaker (E422C) K+ channel (d). In both cases, in 500-nm light the photoswitch is extended, blocking ion conduction, whereas in 380-nm light azobenzene isomerizes to its cis form, retracting the QA and allowing ion conduction. Molecular coordinates from KcsA (Protein Data Bank identifier 2A9H) were drawn using MacPyMol (W.L. DeLano, The PyMOL Molecular Graphics System (2002); http://www.pymol.org). Full Figure and legend (82K)

We assessed the feasibility of the PAL approach using whole-cell patch clamp recording in cells heterologously expressing Shaker K⁺ channels. First, we engineered an appropriately positioned nucleophilic site in Shaker (E422C) to maximize the probability of attachment of the PAL photoswitch to the channel. Addition of AAQ to Shaker E422C photosensitized this channel, enabling regulation of ion flow with light (Fig. 2a). Notably, AAQ also imparted light sensitivity to a Shaker channel lacking the cysteine substitution (Glu422; Fig. 2b) and indeed, conferred a similar degree of light sensitivity on a Shaker channel devoid of extracellular cysteines (data not shown). The fraction of current that could be photoregulated after AAQ treatment (percent photoswitching) was similar for E422C and Glu422 Shaker channels (77 15% and 83 11% respectively, n = 4 cells; Fig. 2c). CAQ and EAQ also photosensitized Glu422 Shaker (data not shown). Hence, PAL molecules can find a covalent attachment site at an appropriate distance from the channel pore, such that light-elicited changes in photoswitch length allow or disallow block by the QA. PAL-modified channels could be photoswitched repeatedly with little decrement in the fraction of current regulated and no apparent photobleaching of the photoswitch (Fig. 2a,b). Thus, PAL molecules can be used to endow persistent light sensitivity to a wild-type Shaker channel, allowing rapid and reversible control of channel function.

Figure 2. Photocontrol of K+ channels expressed in HEK293 cells. (a,b) Voltage-gated currents from Shaker channels after AAQ treatment. Channels containing either an engineered nucleophilic attachment site (E422C; a) or no mutations (b) were exposed to 500-nm and 380-nm light as indicated. Voltage-gated K+ currents were elicited by pulsing from -70 to +30 mV for 250 ms. (c) Percent photoswitching for the indicated channels treated with AAQ (400 M, 15 min). We defined percent photoswitching as the difference between the steady-state current in 380 and 500-nm light, divided by the current in 380-nm light. Current was elicited by stepping from -70 to +30 mV (K+ channels), -80 to 0 mV (Na+ channels), and -40 to +20 mV (Ca2+ channels). Data are average s.d. (n = 4–7 cells for each channel). Full Figure and legend (46K)

If PAL molecules can impart light sensitivity to wild-type Shaker, perhaps they can also act on other QA-sensitive K⁺ channels. We expressed various channels in HEK293 cells and quantified photosensitivity after AAQ treatment (Fig. 2c). Several K⁺ channels became light sensitive, including Kv1.2, Kv1.3, Kv1.4, Kv2.1 and Kv4.2 (percent photoswitching for Kv1.2, 76 16; Kv1.3, 93 9; Kv1.4, 90 13; Kv2.1, 89 5; Kv4.2, 69 4; n = 4 cells each; Fig. 2c). Light sensitivity was less pronounced for Kv3.3 (25 13% photoswitching; n = 5 cells) and BK (24 13% photoswitching; n = 4 cells). In contrast, Kv3.1 exhibited no detectable photosensitivity after AAQ treatment (2 6% photoswitching, n = 6 cells). Moreover, neither voltage-gated Na⁺ channels (Nav1.2) nor voltage-gated Ca²⁺ channels (L-type Cav) became light-sensitive after AAQ treatment (1 5% and -2 5% photoswitching, respectively; n = 4–7 cells).

We next tested whether PAL molecules could photosensitize endogenous K⁺ channels in neurons. Endogenous voltage-gated K⁺ channels set the resting membrane potential and the threshold for triggering action potentials. They also shape the action potential waveform and regulate the propensity to fire repetitively in response to a stimulus. We recorded voltage-gated outward K⁺ currents from cultured hippocampal neurons before and after AAQ application. Steady-state current-voltage curves indicate that AAQ application and exposure to 500-nm light blocked a large fraction of voltage-gated K⁺ currents (Fig. 3a,b; average current blocked 72 9%, n = 5). Photoisomerization of AAQ to its cis configuration with 380-nm light completely relieved K⁺ channel block such that the current-voltage curve overlapped with that before AAQ treatment (Fig. 3a,b; average current recovered under 380-nm light, 117 29%; n = 5). Voltage-gated K⁺ currents were photoswitched to a similar extent in neurons treated with CAQ (data not shown). In contrast, MAQ did not impart light sensitivity to endogenous neuronal K⁺ channels (Fig. 3c; 88 5% and 2 13% photoswitching for AAQ and MAQ respectively; n = 6 each).

Figure 3. Photocontrol of native K+ current in cultured hippocampal neurons. (a) Steady-state current-voltage curves from a voltage-clamped hippocampal pyramidal neuron before (Pre) and after a 15-min application of 300 M AAQ. We measured currents in 380-nm or 500-nm light after AAQ treatment. (b) Voltage-gated currents (elicited by stepping from -70 mV to +30 mV) measured after AAQ treatment were normalized to those measured before AAQ application. Data are average s.d. (n = 5). AAQ-treated channels are completely unblocked by 380-nm light. (c) Percent photoswitching for K+ current in hippocampal neurons treated with AAQ (200 M) or MAQ (250 M). Data are average s.d. (n = 6 for each). Full Figure and legend (26K)

In darkness, the PAL photoswitch relaxes to its trans configuration, blocking PAL-modified K⁺ channels and potentially causing depolarization of the membrane potential. To prevent tonic blockade of K⁺ channels, 380-nm light can be used to keep PAL-modified channels unblocked (Fig. 3a,b). However, because the spontaneous cis to trans isomerization of the photoswitch occurs over many minutes in the dark¹⁰, flashes of 380-nm light (1 s per minute) were sufficient to maintain 94 4% of the K⁺ current unblocked in AAQ-treated neurons (n = 10). Thus, the state of PAL-modified K⁺ channels can be set with the appropriate illumination conditions, preventing tonic K⁺ channel blockade.

Despite having a reactive electrophile and causing K⁺ channel blockade in darkness and 500-nm light, PAL photoswitches did not have obvious deleterious effects on cultured hippocampal neurons. Neurons remained active with a normal resting membrane potential (vehicle, -54 13 mV, n = 10; AAQ, -54 13 mV, n = 23; P > 0.1 unpaired t-test) and normal membrane resistance (vehicle, 77 20 M, n = 10; AAQ, 113 65 M, n = 6; P > 0.1 unpaired t-test) for several hours after PAL treatment. Additionally, there was no significant difference in cell death between neurons treated for 15 min with vehicle alone (5 3%), AAQ (9 3%) or CAQ (8 3%; n = 4–8 fields each, P > 0.05; one-way ANOVA; Supplementary Fig. 1 online). EAQ appeared to be more toxic, and we did not use it in subsequent studies (25 11% cell death). We obtained similar results after treatment for 60 min, four times longer than needed to impart light sensitivity (Supplementary Fig. 1). We also tested neurons up to 6 d after a 15-min treatment with AAQ and found no significant toxicity (3 1% cell death; n = 4 fields, P> 0.05; one-way ANOVA) compared to treatment with vehicle alone (4 1%). Additional experiments showed that AAQ injection into the vitreous humor of the rat eye successfully imparted light sensitivity onto retinal ganglion cells (RGCs) but had no effect on either the histology of the retina or on the rod- and cone-driven light response, as measured in electroretinogram recordings (K.B. and R.H.K.; unpublished observations). Hence, at least for our treatment conditions, AAQ does not appear toxic to neurons in vitro or in vivo.

Figure 4. Photocontrol of neuronal firing. (a,b) Current clamp recordings from hippocampal neurons treated with AAQ and exposed to alternating 380-nm and 500-nm light as indicated to control action potential firing (a), to a 200-ms flash of 500-nm light followed by either 380-nm light to trigger a transient burst of spikes (b; left) or followed by darkness to trigger sustained activity (b; right). Depolarizing current was injected throughout. (c) Current clamp recordings of single action potentials in an AAQ-treated neuron illuminated with 500-nm light (green) or 380-nm light (violet). Action potentials were induced with 500 ms depolarizing current injection (120 pA in 380-nm light, 100 pA in 500-nm light). Full Figure and legend (36K)

Figure 5. Modulation of neuronal excitability with light. (a) After PAL treatment identical current pulses (Iinj 250 pA, 50 ms, 5 Hz) that did not elicit changes in membrane voltage (Vm) sufficient to trigger spikes in 380-nm light (top), reliably elicited spikes in 500-nm light (bottom). (b) Effect of different wavelengths of light on voltage-gated K+ channel blockade and action potential threshold in neurons exposed to light of increasing wavelength. For each wavelength, the percent maximal block of voltage-gated K+ current (n = 5 cells) was compared to the current injection threshold required to fire a spike (n = 6 cells). (c) Effect of different wavelengths of light on spike frequency adaptation. Current injection (300 pA, 1 s) in neurons treated with PAL was performed during exposure to 380-, 400-, 420- or 500-nm light, eliciting different firing patterns. (d) Relationship between illumination wavelength and average spike frequency during depolarizing pulses (n = 9 cells). (e) Effect of illumination wavelength on firing threshold and sensitivity to progressively greater current injections in neurons treated with PAL. Currents were normalized to the maximal amount of current injected in each cell. Current injections lasted 500 ms and ranged from 15 to 360 pA (n = 6 cells). All data are means s.d. Full Figure and legend (70K)

Channel modification by AAQ is dependent on the presence of a nucleophile at the correct distance from the QA binding site. We showed that covalent attachment occurs when cells are treated in the dark with AAQ in its extended trans form. However, the reactive moiety of PAL will encounter different amino acids when the photoswitch is in its shorter cis configuration. To test whether AAQ can attach to K⁺ channels in its cis form, we added AAQ while illuminating a subpopulation of neurons with 380-nm light (Fig. 6a). The remaining neurons were kept in darkness and thus were exposed to AAQ in its trans form. Neurons illuminated with 380-nm light during PAL treatment showed little photosensitivity, whereas those kept in darkness exhibited light-regulated K⁺ currents (Fig. 6b–d; treatment in 380-nm light, 5 5% photoswitching, n = 10; treatment in darkness, 54 16% photoswitching, n = 9). The magnitude of the unblocked K⁺ current in the two groups of cells was not significantly different (3,515 1,022 pA, n = 9 and 4,355 1,466 pA, respectively, n = 10; P > 0.1, Student's unpaired t-test). Hence, photosensitization of K⁺ channels by PAL is itself sensitive to light. This feature can be exploited to pre-program specific cells to become light-sensitive by using selective illumination during PAL treatment.

Figure 6. Local illumination during PAL treatment imprints photosensitivity onto specific neurons. (a) A hippocampal neuronal culture was uniformly exposed to 300 M AAQ. During treatment, the microscope objective was used to illuminate a subpopulation of neurons with 380-nm light while the remaining neurons were kept in the dark. After treatment, AAQ was replaced with extracellular solution, whole-cell recordings were obtained, and photoresponsiveness was tested by applying alternating flashes of 380-and 500-nm light. (b,c) Photoresponsiveness of neurons treated with AAQ either in 380-nm light (b) or in darkness (c). Currents were elicited by stepping from -70 mV to +30 mV for 250 ms every 2 s while illuminating with 380-nm or 500-nm light. Recordings in b and c were from neurons on the same coverslip. (d) Summary data showing average percent photoswitching of neurons treated with AAQ in 380-nm light or the dark. Data are averages s.d. (n = 9–10 neurons). Full Figure and legend (45K)

Figure 7. Photocontrol of action potential firing in intact neural circuits. (a) Rat cerebellar slices were treated with PAL, and a loose-patch recording of spontaneous spiking was obtained from a cerebellar basket cell during alternating 360-nm and 500-nm light. Basket cells were synaptically isolated from other neurons by using GABA and glutamate receptor antagonists. (b) Simultaneous loose-patch recordings of bursting activity from the left and right heart interneurons (HNL and HNR) in the medicinal leech heartbeat central pattern generator after PAL treatment and subsequent illumination with 380-nm and 500-nm light. (c) Loose-patch recording of PAL-mediated photoresponses in a rat RGC from a flat-mounted retina under alternating illumination as in b. Full Figure and legend (61K)

The installation of light sensitivity on neurons may enable the artificial input of information downstream from sites of damage or degeneration. For example, the loss of visual function caused by degeneration of rods and cones could be alleviated by treatments that impart light sensitivity on downstream neurons that are normally light insensitive. We tested whether PAL treatment could impart light sensitivity on RGCs, which relay information from the retina to the brain. Loose-patch recordings obtained from RGCs after AAQ treatment showed that their firing increased in 500-nm light and decreased in 380-nm light (Fig. 7c), owing to the block and unblock of AAQ-modified K⁺ channels. We observed robust photocontrol of firing in 15/23 RGCs in AAQ-treated retina and 0/10 RGCs in non–AAQ-treated retina. The retina contains a small fraction of RGCs (2.5%) that express melanopsin and are intrinsically photosensitive²⁷, but it is unlikely that these cells account for the AAQ results reported here.

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We grew HEK293 cells in DMEM containing 5% FBS. For HEK293T cells, we also included 500 g/ml G-418. For electrophysiology studies, we plated cells at 12 10³ cells/cm² on poly(L-lysine)–coated coverslips and transfected the cells using the calcium phosphate method²⁹. The Shaker H4 used here contained a deletion of amino acids 6–46 to minimize fast inactivation³⁰. We transfected plasmids encoding Kv4.2 and KChIP3 at a 4:1 ratio³¹. We recorded K⁺ channel currents 24–48 h after transfection of HEK293 cells and Nav1.2 currents 96 h after transfection of HEK293T cells. We recorded currents for L-type Ca²⁺ channels from GH3 cells grown in F-12K containing 15% horse serum and 2.5% FBS, 48 h after plating. We prepared hippocampal neurons from neonatal rats according to standard procedures³², plated them at 50 10³ cells/cm² on poly(L-lysine)–coated coverslips and grew them in minimum essential medium containing 5% FBS, 20 mM glucose, B27 (Invitrogen), glutamine and Mito+ Serum Extender (BD Biosciences). We recorded currents 14–25 d after plating. Animal care and experimental protocols were approved by the University of California Berkeley Animal Care and Use Committee.

We incubated cells at 37 °C in the dark for 15 min with 200–300 M (for hippocampal neurons) or 400 M (for HEK293, HEK293T and GH3 cells) AAQ diluted in bath solution. We treated the cerebellar slices at room temperature in the dark with 200 M AAQ for 8 min, and we treated retinas and leech ganglia with 150 M and 400 M AAQ, respectively.

For illumination for photoswitching, we used a xenon lamp (175 W) with narrow band-pass filters (380BP10 and 500BP5). We measured light output using a handheld Newport meter (840-C model). At the back of the objective, light output was 0.3 mW/cm² for 380-nm light and 2.5 mW/cm² for 500-nm light. When measured through a 40 objective and normalized to the focal area at the specimen plane, light output was 0.5 mW/mm² and 3.5 mW/mm² for 380-nm and 500-nm light, respectively. For some experiments, we used a monochromator (Polychrome V; TILL Photonics) for illumination.

All data reported are averages s.d.

We recorded currents in the whole-cell patch clamp configuration using pipettes with 3–5 M resistance. To elicit voltage-gated K⁺ currents from neurons and HEK293 cells, we set holding potential to -70 mV and stepped to +30 mV for 250 ms. To elicit voltage-gated Na⁺ currents, we held HEK293T cells expressing Nav1.2 at -80 mV and stepped to 0 mV for 250 ms. We recorded L-type Ca²⁺ currents in GH3 cells and elicited the currents by stepping to +20 mV for 200 ms from a holding potential of -40 mV. Bath and intracellular solutions compositions are available in Supplementary Methods.

We prepared parasagittal cerebellar slices (300 m) from postnatal day 14–20 rats using standard techniques approved by the University of California Los Angeles Animal Care Committee. After sectioning, we incubated slices for 30 min at 35 °C in aCSF (composition in Supplementary Methods) then brought them to room temperature for PAL treatment and recording. We made loose-patch recordings in the presence of 6,7-dinitroquinoxaline-2,3-[1H,4H]-dione, gabazine and [RS]-3-[2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid using pipettes with 3–5 M resistance. The depth of recorded basket cells was approximately 25–30 m. We dissected retinas from postnatal day 50–60 rats under continuous flow of oxygenated saline solution (composition in Supplementary Methods). We treated dissected retinas at 37 °C with 20 U/ml cysteine-activated papain for 8–20 min, then washed them with saline containing 10 mg/ml BSA and 10 mg/ml trypsin inhibitor followed by PAL treatment and recording using the loose-patch configuration. Dissections and recordings from leech ganglia were conducted as described previously³³. We obtained extracellular signals from HN cells using the loose-patch configuration.

Detailed descriptions of the chemical syntheses and the electrophysiology solutions are available in Supplementary Methods. All data reported are averages s.d.

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