Table 1.
Composition of bath and pipette solutions in experiments with ND7/23 cells and neurons.
Fig 1.
Basic characterization of KR2 photocurrent in ND7/23 cells.
Representative photocurrent at 0 mV in the absence (A) and presence (B) of 110 mM NaCl in intracellular solution while the bath solution contained 140 mM NaCl. pHs were adjusted at 7.4 for both solutions. See Table for more details. 530 nm light (25 mW/mm2) was illuminated for 200 ms as the green bar indicates. C and D, light power dependency on the peaks (Ip) and steady state (Iss) photocurrent, in the absence and presence of NaCl intracellular solution, respectively (n = 7). E and F, Current-voltage relation (I/V plot) for the peak current (n = 12, 9). The currents were normalized to the value at 0 mV. G and H, Current-voltage relation (I/V plot) for the steady state component. The currents were normalized to the value at 0 mV (n = 12, 9).
Fig 2.
Effect of intracellular pH (pHi).
Photocurrent amplitude at 0 mV under three pHi conditions (7.4, 8.0 and 9.0) in the absence (A) and presence (B) of 110 mM NaCl inside (n = 6–14, *p<0.05). The bath solution contained 140 mM NaCl at pH 7.4. See Table for more details. Gray bar: peak current (Ip); white bar: steady state current (Iss). Off kinetics value (τoff LED) under 3 pHi conditions in the absence (C) and presence (D) of 110 mM NaCl in intracellular solutions. E, Representative KR2 photocurrent in double pulse experiment with various dark periods at 0 mV. LED light at 530 nm was applied as indicated by green lines below the photocurrent traces. The dark interval between the two light pulses was prolonged by 5 ms. Photocurrents traces are overlaid in different gray scales. The second peak amplitude was recovered as the dark interval was prolonged while the steady state current remained unchanged. The inter-sweep dark period was always set at 2.0 s to guarantee completed dark adaption. The intracellular solution contained 110 mM NaCl at pH 7.4 while the bath solution contained 140 mM NaCl at pH 7.4. D, Time course of the second peak recovery (n = 6–22) (F and G). The second peak ratio (Ip2/Ip1) was plotted as a function of the dark period (ms). F, The measurement was performed at three different pHi in the absence of NaCl in the intracellular solutions. G, the same experiment as in F but in the presence of 140 mM NaCl inside the cells (n = 6–8). H and I, kinetic values obtained from F and G (* p<0.05) (τRecovery). The recovery time course in F and G was plotted with a single exponential function.
Fig 3.
Effect of intracellular Na+ concentration.
A, The recovery kinetics of the second peak photocurrent in the presence of various NaCl concentrations at pH 7.4 in intracellular solution (n = 6–16). See Table for more details. The same recordings shown in Fig 2E were performed to assess the effect of Na+. B, Kinetic parameters (τRecovery) obtained from A (* p<0.05, ** p<0.01). C, Photocurrent kinetics after shutting the LED light (τoffLED) was determined under various NaCl concentrations inside the cell. D, Photocurrent amplitude in various NaCl concentrations inside the cell while the bath solution contained 140 mM NaCl (n = 10–14, * p<0.05).
Fig 4.
Effect of additional blue light (probing M intermediate).
A and B, Representative photocurrent traces in the absence (A) and presence (B) of intracellular NaCl (110 mM) at pH 7.4 while the bath solution contained 140 mM NaCl a pH 7.4. The membrane voltage was clamped at 0 mV. Blue LED (470 nm at 54 mW/mm2) was illuminated on top of a green LED illumination (530 nm at 4.6 mW/mm2), as shown by colored bars under the current traces. C and D, The recordings as A and B with a green light (530 nm at 0.49 mW/mm2) in the absence (C) and presence (D) of intracellular NaCl. The inset in C shows enlarged traces upon blue light illumination at different membrane voltages. E-H, Current-voltage relation (I/V plot) of the photocurrent during additional blue light illumination (n = 6). The peak component Ip470 and the steady state level (Is470) were plotted. E and G, in the absence of intracellular NaCl. F and H, in the presence of 110 mM NaCl. E and F, Strong green light (6.6 mW/mm2). G and H, Weak green light (0.49 mW/mm2).
Fig 5.
Single photocycle ion transport.
Photocurrent of KR2 evoked by a 5 ns flash laser (532 nm) at 0 mV. The recording was performed in the absence (A) and presence (B) of intracellular Na+ (110 mM) at pHi 7.4, while bath solution contained 140 mM NaCl at pH 7.4. C, Off-kinetics values obtained from a flash laser (τoff Laser), a green LED (τoff LED) (n = 7–16, * p<0.05, ** p<0.01). Open bar, in the absence of intracellular Na+. Gray bar, in the presence of 110 mM NaCl inside.
Fig 6.
A, Representative traces of action potentials by current injection (200 pA) as shown below the trace in a KR2-expressing cultured neuron. Solutions used were listed in Table. B, Evoked spikes were suppressed by green light illumination as indicated by a green bar in a KR2-expressing neuron. C and D, Spike frequency at various injected currents are compared in KR2-expressing neurons. Number of spikes in darkness (Pre-illumination), upon illumination (During-illumination) and again in darkness (Post-illumination) are indicated. C, no illumination in “During” shown by white bars. D, illumination in “During” shown by green bars. n = 6, 7. E, light power dependency on neuronal silencing (n = 6). Spike frequency is shown at various light intensities. The neuron was excited by 150 pA current injections.
Fig 7.
Reaction scheme of the photo/transport cycle of KR2.
A, The cycle is initiated by green light absorption of the Dark state to form M-intermediate. The M-intermediate decays in to the dark state with a time constant of 14 ms (τoff LED). KR2 transport H+ indicated in blue arrows. Blue light illumination triggers a photoisomerization of M-intermediate, which result in a no-transport cycle indicated in a dotted line. B, Similar cycle can be proposed for Na+ transport with time constant of 9.7 ms (τoff LED). The no-pump cycle by blue light (black arrow) is favored in the presence of Na+ compared to the absence of Na+, because M-intermediate is more stable according to the result in Fig 4.