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Figure 1. Experimental strategy. The purpose of our study was to elicit light-induced APs not by coexpression of wild type ChR2 with voltage-gated Na+ channels, but by coupling ChR2 either directly to Nav1.5 (ChR2-Nav1.5, Nav1.5-ChR2) or to the accessory β1 subunit (β1-ChR2, β1-ChR2-ChR2). The seven transmembrane regions of ChR2 (blue), the four large domains of voltage-gated Na+ channels (red), the β1 subunit (orange), and the membrane-spanning region of the human H+/K+ATPase (gray) are illustrated. In Nav1.5-ChR2, β1-ChR2, and β1-ChR2-ChR2, peptide [GGGS]3 was incorporated into all linker regions (symbolized by a black line). For detailed amino acid composition of the constructs, see Materials and methods.
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Figure 2. Electrophysiological test for functional expression of ChR-Nav1.5 and Nav1.5-ChR2 in Xenopus oocytes.
(A) Representative whole-cell currents generated by wild type Nav1.5, ChR2-Nav1.5, and Nav1.5-ChR2 at test potentials between −45 and +20 mV. Injected amount of cRNA per oocyte: 0.1 ng Nav1.5, 10 ng ChR2-Nav1.5, and 10 ng Nav1.5-ChR2. Peak current density for Nav1.5 was 1.56 ± 0.1 µA (n = 10). Current reduction was similar in ChR2-Nav1.5 and Nav1.5-ChR2 (0.59 ± 0.08 µA with n = 21 versus 0.71 ± 0.07 µA with n = 27, respectively; P < 0.05 versus Nav1.5). (B) None of the ChR2 fusions responded to a 1-s blue-light pulse. Injected amount of cRNA per oocyte: 0.2 ng ChR2, 10 ng ChR2-Nav1.5, and 10 ng Nav1.5-ChR2.
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Figure 3. Whole-cell currents through ChR2, β1-ChR2, and β1-ChR2-ChR2 channels.
(A) Linkage of the C-terminus of the β1 subunit to the N-terminus of ChR2 resulted in typical light-induced inward currents in the voltage-clamp mode. The plateau current relative to the transient current was similar for all three channels (52.9 ± 7.6% in ChR2, 42.8 ± 8.2% in β1-ChR2, and 42.0 ± 10.8% in β1-ChR2-ChR2). (B) Currents were significantly reduced when injecting 10 ng cRNA per oocyte. Notably, coupling of a second ChR2 did not alter expression (possible reasons are discussed in the text). Number of measurements were n = 27 (ChR2), n = 20 (β1-ChR2), and n = 21 (β1-ChR2-ChR2; *, P < 0.05 versus ChR2). Error bars represent SEM.
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Figure 4. Comparison of membrane fluorescence and photocurrents in Xenopus oocytes expressing ChR2-YFP and β1-ChR2-YFP.
(A) Representative fluorescence images of Xenopus oocytes. Noninjected control oocytes showed only faint background fluorescence. Scale bars: 0.25 mm. (B) Normalized fluorescence intensities. Data points are from three different oocyte batches with n = 22 for ChR2-YFP and n = 19 for β1-ChR2-YFP (*, P < 0.05 versus ChR2-YFP). (C) Representative whole-cell currents through ChR2-YFP and β1-ChR2-YFP. (D) Peak current amplitudes. Number of measurements were n = 27 for ChR2-YFP and n = 26 for β1-ChR2-YFP (*, P < 0.05 versus ChR2-YFP). Four different oocyte batches were used. Error bars represent SEM.
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Figure 5. Representative Nav1.4 current traces at a test potential of −10 mV in Xenopus oocytes. Coexpression of β1 resulted in a faster decay of macroscopic Na+ currents through Nav1.4 channels. To better illustrate the effect on inactivation, we selected whole-cell currents with similar amplitude. Current traces were fitted (red) using a biexponential function: I = Af × exp(−t/τf) + As × exp(−t/τs), where τf and τs are the fast and slow time constants, and Af and As are the corresponding amplitudes. Both fast and slow time constants, τf and τs, were significantly shorter. This well-known β1 property remained preserved in β1-ChR2 and β1-ChR2-ChR2 and was not obtained when coexpressing ChR2 (top). Values for τf, Af, τs, and As are from at least six different measurements (*, significantly different versus Nav1.4, P < 0.05). This typical acceleration of inactivation was also obtained at all other test pulses.
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Figure 6. Acceleration of recovery from inactivation of Nav1.4 by β1, β1-ChR2, and β1-ChR2-ChR2. The respective voltage protocol is shown on the right. During the first pulse (500 ms at −10 mV), Nav1.4 channels were activated; subsequently they accumulated in the inactivated state. During the time interval Δt, channels were allowed to recover at −120 mV. The second test pulse to −20 mV was used to determine the respective fraction of non-inactivated channels. Coexpression of β1 as well as both ChR2-coupled β1 subunits accelerated this recovery process. Data points are from two oocyte batches and four to eight different recordings. For statistics, see Table 1. Error bars represent SEM.
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Figure 7. Light-induced APs in Xenopus oocytes coexpressing Nav1.4 and β1-ChR2. All recordings shown were performed on a single oocyte. Light pulses are illustrated as blue bars. (A) Current-clamp recording. The membrane potential was set to −80 mV, and the change of membrane voltage was followed on a 25-ms light pulse delivered to the whole oocyte. For AP variability and for detailed statistics, see Fig. S1 and Table S1. (B) Corresponding whole-cell current through β1-ChR2 channels recorded in the voltage-clamp mode. Pulse duration was 1 s. (C) Nav1.4 whole-cell current of the same oocyte recorded in the voltage-clamp mode (holding potential, −120 mV; test pulse, −25 mV). (D) Current-clamp recording in the presence of 400 nM TTX (red). The remaining depolarization was due to ChR2 activity. Washout restored the fast upstroke to nearly +20 mV (black). (E) TTX did not affect β1-ChR2 activity (voltage-clamp recording as in B). (F) Nav1.4 channels were completely blocked in the presence of 400 nM TTX (voltage-clamp mode as in C). Black line shows the Na+ current after washout of TTX.
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Figure S1. Light-induced APs in Xenopus oocytes using the skeletal muscle Na+ channel subtype Nav1.4.
(A) Thirteen superimposed current-clamp recordings in oocytes expressing Nav1.4 and β1-ChR2. (B) Eleven superimposed current-clamp recordings in oocytes expressing Nav1.4, β1-ChR2, and Kv2.1. Coexpression of the voltage-gated K+ channel led to AP shortening and a less pronounced variability in AP duration and shape. For detailed statistics, see Table S1. The blue-light flash is indicated by an arrow.
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Figure 8. Light-induced changes of membrane potential and membrane current upon coexpression of Nav1.4 and β1-ChR2 in Xenopus oocytes.
(A) Current-clamp and voltage-clamp recording in the absence of TTX. The membrane potential was set to −80 mV, and both the change of membrane voltage and the inward current were followed on a 25-ms light pulse delivered to the whole oocyte. (B) Current-clamp and voltage-clamp recording in the presence of 400 nM TTX (same oocyte as in A). The toxin did not reduce the light-triggered inward current recorded at −80 mV. Pulsing from −120 to −35 mV in the voltage-clamp mode revealed that the toxin blocked >99% of Nav1.4 channels, similarly as shown in Fig. 7.
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Figure 9. Acceleration of AP repolarization by Kv2.1. Recordings were performed on a single oocyte coexpressing Nav1.4, β1-ChR2, and Kv2.1. (A) Current-clamp recording (pulse duration 25 ms). For statistics on AP shortening by Kv2.1, see Table S1. (B) Corresponding whole-cell current through β1-ChR2 channels recorded in the voltage-clamp mode (pulse duration 1 s). (C) Nav1.4 whole-cell current of the same oocyte recorded in the voltage-clamp mode (test pulse −25 mV). (D) Current-clamp recording at 400 nM TTX (red). Washout restored the AP (black). (E) Currents through β1-ChR2 in the absence and presence of TTX (pulse duration 1 s). (F) Block of Nav1.4 channels by 400 nM TTX (red). Washout completely restored the voltage-gated Na+ current. (G) Corresponding Kv2.1 current (voltage-clamp mode). Test pulses are indicated. The overlapping Na+ current is not visible due to the presence of 400 nM TTX. The amount of coinjected Kv2.1 cRNA was 50 pg per oocyte. The magnitude of the injected current required to set the initial membrane potential to −80 mV was not altered by Kv2.1 coexpression.
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Figure 10. Effect of the nonspecific K+ channel blocker TEA on light-triggered APs in Xenopus oocytes. Recordings were performed on a single oocyte expressing Nav1.4, β1-ChR2, and Kv2.1. Duration of the light pulse was 25 ms. (A) Current-clamp recording in the absence and presence of 50 mM TEA. TEA reduced the upstroke velocity, which was due to a partial block of Nav1.4 channels, and caused a deceleration of AP repolarization, which was due to a reduction of the Kv2.1 current. The early depolarization phase to nearly −55 mV remained unchanged, because β1-ChR2 currents were not affected by TEA. Corresponding light-triggered current values were as follows: Ipeak = 581 ± 212 nA, Iplateau = 279 ± 95 nA (without TEA), and Ipeak = 605 ± 224 nA, Iplateau = 307 ± 109 nA (50 mM TEA; P > 0.2 with n = 10). (B) Corresponding Nav1.4 currents. The test pulse was −10 mV. At 50 mM TEA, Nav1.4 was partially blocked (red). (C) Corresponding Kv2.1 currents. At 50 mM TEA, >75% of Kv2.1 channels were blocked, causing the pronounced AP prolongation seen in A.
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Figure 11. Effect of light pulse duration in two different oocytes coexpressing Nav1.4, β1-ChR2, and Kv2.1 on the membrane potential and on the corresponding photocurrent. Duration of the light flashes was 25, 10, and 2 ms. Amounts of coinjected cRNA per oocyte were 10 ng Nav1.4, 2.75 ng β1-ChR2, and 50 pg Kv2.1. (A) The 25-ms pulse induced a peak photocurrent of 0.51 µA, which led to successful AP triggering in this oocyte (black line). In contrast, 10 and 2 ms were too short to activate a sufficient number of β1-ChR2 channels, so the threshold potential was not achieved (red and purple lines, respectively). Light-induced peak currents were 0.44 and 0.14 µA, respectively. (B) Blue-light pulses induced larger photocurrents, indicating a higher β1-ChR2 expression level. The 25-ms pulse resulted in a peak photocurrent of 0.70 µA and produced a typical AP (black). The short 2-ms pulse was not sufficient to elicit an AP. The corresponding light-induced current of 0.31 µA was larger than in A, and consequently, the subthreshold depolarization was more pronounced (compare purple curves in upper graphs in A and B). Application of an intermediate pulse (10 ms, red) resulted in a peak photocurrent of 0.67 µA, and in above-threshold activation. However, depolarization was delayed compared with the longer light pulse of 25 ms. In both oocytes, the membrane resistance as well as the Nav1.4 current at −30 mV were very similar (0.59 MΩ and 16.6 µA in A and 0.5 MΩ and 16.9 µA in B, respectively). K+ currents at +70 mV were 6.4 µA (A) and 8.0 µA (B).
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Figure 12. Light-induced APs in Xenopus oocytes coexpressing β1-ChR2 and Kv2.1 with either Nav1.2 or Nav1.5.
(A and C) Current-clamp recordings. For detailed statistics on the effect of Kv2.1 on Nav1.5-mediated APs, see Fig. S2 and Table S2. (B and D) Corresponding Na+ currents in the absence and presence of TTX. Similarly to Nav1.4, neuronal Nav1.2 channels belong to the TTX-sensitive Na+ channels; they can be blocked at nanomolar TTX concentrations. Cardiac Nav1.5 channels are TTX resistant, with IC50 values >1 µM. The peak and plateau currents through β1-ChR2 were Ipeak = 768 nA, Iplateau = 193 nA (Nav1.2-expressing cell in A and B), and Ipeak = 957 nA, Iplateau = 193 nA (Nav1.5-expressing cell in C and D). The K+ currents through Kv2.1 were I+70 mV = 14.3 µA (Nav1.2-expressing cell, A and B), and I+70 mV = 30.9 µA (Nav1.5-expressing cell, C and D). Duration of the light pulse was 25 ms.
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Figure S2. Light-induced APs in Xenopus oocytes using the cardiac Na+ channel Nav1.5.
(A) Nine superimposed current-clamp recordings in oocytes expressing Nav1.5 and β1-ChR2. (B) Seven superimposed current-clamp recordings in oocytes expressing Nav1.5, β1-ChR2, and Kv2.1. Similarly, as observed for Nav1.4-injected oocytes, coexpression of Kv2.1 led to a pronounced AP shortening. For detailed statistics, see Table S2. The blue-light flash is indicated by an arrow.
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Figure 13. Repetitive firing of light-triggered APs in Xenopus oocytes coexpressing either neuronal Nav1.2, skeletal muscle Nav1.4, or cardiac Nav1.5 with β1-ChR2 and Kv2.1. The membrane potential was set to −80 mV (Nav1.2 or Nav1.5) or −85 mV (Nav1.4), and the change of membrane voltage was followed in the current-clamp mode on 50-ms light pulses every 2 s.
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