Precise spatiotemporal control of voltage-gated sodium channels by photocaged saxitoxin

Abstract

Here we report the pharmacologic blockade of voltage-gated sodium ion channels (Na_Vs) by a synthetic saxitoxin derivative affixed to a photocleavable protecting group. We demonstrate that a functionalized saxitoxin (STX-eac) enables exquisite spatiotemporal control of Na_Vs to interrupt action potentials in dissociated neurons and nerve fiber bundles. The photo-uncaged inhibitor (STX-ea) is a nanomolar potent, reversible binder of Na_Vs. We use STX-eac to reveal differential susceptibility of myelinated and unmyelinated axons in the corpus callosum to Na_V-dependent alterations in action potential propagation, with unmyelinated axons preferentially showing reduced action potential fidelity under conditions of partial Na_V block. These results validate STX-eac as a high precision tool for robust photocontrol of neuronal excitability and action potential generation.

Fig. 1. Caging STX-ea 1 with photo-protecting groups.

a Synthesis and selective carbamoylation of STX-ea 1. b Photocaged derivatives of STX-ea.

Fig. 2. Photochemical release of STX-ea 1 results in a block of Na_V1.2 CHO.

a Electrophysiological characterization of photocaged STXs against Na_V1.2 CHO. IC₅₀s, Hill coefficients: 1 = 14.4 ± 0.3 nM, –0.94 ± 0.02; 2 = 27.0 ± 1.4 nM, –0.94 ± 0.05; 3 = 55.5 ± 2.1 nM, –1.01 ± 0.04; 4 = 307.5 ± 16.6 nM, –1.37 ± 0.09; 5 = 1003.8 ± 42.2 nM, –1.00 ± 0.04. Data represent mean ± s.e.m. (for compound 1 [2, 5, 10 nM], n = 6, [20, 50, 100 nM], n = 7; 2, n = 3; 3, n = 6; 4, n = 6; 5, n = 5). b Electrophysiological characterization and laser uncaging of 5 against Na_V1.2 CHO. Initial IC₅₀ in blue; apparent IC₅₀ following laser scan 1 in red (30.4 ±  3.6 nM) and after laser scan 5 in purple (10.4 ± 2.0 nM). Data represent mean ± s.e.m. (n = 5). c Representative trace depicting uncaging of 100 nM 5 against Na_V1.2 CHO. Traces collected in the order: Start, 100 nM 5, 0 s, 2 s, 4 s. Laser applied immediately prior to 10 ms, 0 mV voltage step (trace 0 s). d Time course of uncaging of 100 nM 5 against Na_V1.2 CHO pulsed from –100 mV to 0 mV at 2.5 Hz. The laser was applied at t = 0 s. Data were subjected to exponential regression yielding τ = 2.3 ± 0.2 s (half-life 1.6 ± 0.1 s), R² = 0.9056. Data represent mean ± s.e.m. (n = 4). n = number of biologically independent cells.

Fig. 3. Uncaging of STX-eac 5 results in fast, concentration-dependent Na_V block and inhibition of APs.

a Electrophysiological characterization of photocaged STXs against hippocampal neurons DIV 6–8. IC₅₀s, Hill Coefficients: 1 = 14.1 ± 0.8 nM, –0.86 ± 0.04; STX-eac 5 = 2148.4 ± 209.9 nM, –0.83 ± 0.07. Apparent IC₅₀, Hill Coefficient: 5 (uncaged) = 106.0 ± 17.4 nM, –0.71 ± 0.11. Data represent mean ± s.e.m. (for compound 1 [2 nM], n = 4; [5, 10, 20, 50, 100 nM], n = 5. For compound 5 [100 nM], n = 12; [200 nM], n = 10; [500 nM], n = 13; [1 µM], n = 12; [2 µM, 5 µM], n = 4. For compound 5 (uncaged) [100 nM], n = 4; [200 nM], n = 6; [500 nM], n = 5; [1 µM], n = 4.). b Representative trace depicting uncaging of 200 nM 5 against hippocampal neurons DIV 6–8. Traces collected in the order: Start, 200 nM 5, 0 s, 2 s, 4 s. Laser applied immediately prior to 10 ms, 0 mV voltage step (trace 0 s). c Comparison between current-clamp (DIV 9–13, right axis, black) and voltage-clamp (DIV 6–8, left axis, gray) data collected pre- and post-uncaging at various concentrations of 5. T, toxin applied; TL, toxin and laser applied. Statistics calculated for current-clamp data. ns P > 0.5, ***P < 0.001, one-way ANOVA with Tukey’s correction, mean ± s.e.m. T(100 nM) vs. T(200 nM), P = 0.7727; T(100 nM) vs. T(500 nM), P = 1.34 × 10^–4; T(200 nM) vs. T(500 nM), P = 8.46 × 10^–4; T(500 nM) vs. TL(100 nM), P = 0.9997. (For T(100 nM), n = 9; T(200 nM), n = 19; T(500 nM), n = 5; TL(100 nM), n = 9; TL(200 nM), n = 19; TL(500 nM), n = 5.) n = number of biologically independent cells.

Fig. 4. Uncaging of 200 nM STX-eac 5 shows reversible inhibition of AP firing.

a Representative traces depicting initial (I), laser applied (L), 200 nM toxin 5 applied (T), 200 nM toxin 5 and laser applied (TL), and recovered (R) after wash-off AP trains evoked by 500 ms, 50–150 pA current injections into hippocampal neurons DIV 9–13. Data taken from replicate current step 2 vis-à-vis (b). b Heatmap summary of data described in a color-coded by the number of action potentials per step (four replicate current steps at 0.25 Hz per condition, n = 19). c Equilibrated normalized action potential firing rate (i.e., over current steps 2–4) pre- and post-laser induced uncaging of 5. ns P > 0.5, ****P < 0.0001, one-way ANOVA with Tukey’s correction, mean ± s.e.m. I vs. L, P = 0.9503; I vs. T, P = 0.4131; L vs. T, P = 0.7437; I vs. TL, P = 2.7 × 10^–14; L vs. TL, P = 9.69 × 10^–12; T vs. TL, P = 2.38 × 10^–8. (n = 19). n = number of biologically independent cells.

Fig. 5. Uncaging STX-eac 5 differentially affects myelinated vs. unmyelinated callosal fiber transmission.

a Experimental design of corpus callosum preparation. On the left, a representative picture of an experiment. On the right, a corresponding schematic representation, HC stands for the hippocampus; cc for corpus callosum. b Schematic representation of callosal N1 and N2 features. N1 is the fastest component and corresponds mainly to myelinated axon fibers. N2 is slower and corresponds mainly to unmyelinated axon fibers. c Example of LFP and CSD signals for two different concentrations of 5, 250 nM (upper panel) and 500 nM (lower panel). Black traces represent signals before uncaging, gray traces represent signals after uncaging by 500ms UV pulse light delivery. Only the first seven channels closest to the stimulating electrode are represented. The expanded timescale of the first CSD channel is represented on the right. For 250 nM 5, a reduction of amplitude and a delay of the peak time can be observed after uncaging for N2. Only a reduction of amplitude can be observed for N1; peak time was not affected. For 500 nM 5, a loss of N1 and N2 signals can be observed after light delivery. d Scatter plot of the effect of 100 nM bath application of 1 in blue and of the effect of uncaging of 100, 250, and 500 nM 5 on N1 (left graph) and N2 (right graph) amplitude. e Scatter plot of the effect 100 nM bath application of 1 in blue and of uncaging of 100, 250, and 500 nM 5 on the N1 (left) and N2 (middle) peak time. Histograms (on the right) represent the incidence of complete block for N1 (black) and N2 (red) for the three different concentrations of 5, 100, 250, and 500 nM. Increases in peak timing were observed with 100 nM 1 and 500 nM 5 for N1 and with 100 nM 1, 100 nM and 250 nM 5 for N2. Notice that 30% of the slices have a complete block of N2 at 500 nM, obscuring any potential difference in latency. d, e Two-tailed Wilcoxon test (n = 8, 7, 5, and 8, respectively, for 100 nM 1, 100 nM, 250 nM, and 500 nM 5). P values less than 0.05 are highlighted in red. Each pair of connected dots represents the results of a single slice before (filled circle) and after (open circle) light application. For each condition mean ± s.e.m. are represented on each side of the connected dots.