Jerng HH et al. (2021), Light-regulated voltage-gated potassium channel...

XB-ART-57945

PLoS One 2021 Mar 03;163:e0248688. doi: 10.1371/journal.pone.0248688.

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Light-regulated voltage-gated potassium channels for acute interrogation of channel function in neurons and behavior.

Jerng HH , Patel JM , Khan TA , Arenkiel BR , Pfaffinger PJ .

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Voltage-gated potassium (Kv) channels regulate the membrane potential and conductance of excitable cells to control the firing rate and waveform of action potentials. Even though Kv channels have been intensely studied for over 70 year, surprisingly little is known about how specific channels expressed in various neurons and their functional properties impact neuronal network activity and behavior in vivo. Although many in vivo genetic manipulations of ion channels have been tried, interpretation of these results is complicated by powerful homeostatic plasticity mechanisms that act to maintain function following perturbations in excitability. To better understand how Kv channels shape network function and behavior, we have developed a novel optogenetic technology to acutely regulate Kv channel expression with light by fusing the light-sensitive LOV domain of Vaucheria frigida Aureochrome 1 to the N-terminus of the Kv1 subunit protein to make an Opto-Kv1 channel. Recording of Opto-Kv1 channels expressed in Xenopus oocytes, mammalian cells, and neurons show that blue light strongly induces the current expression of Opto-Kv1 channels in all systems tested. We also find that an Opto-Kv1 construct containing a dominant-negative pore mutation (Opto-Kv1(V400D)) can be used to down-regulate Kv1 currents in a blue light-dependent manner. Finally, to determine whether Opto-Kv1 channels can elicit light-dependent behavioral effect in vivo, we targeted Opto-Kv1 (V400D) expression to Kv1.3-expressing mitral cells of the olfactory bulb in mice. Exposure of the bulb to blue light for 2-3 hours produced a significant increase in sensitivity to novel odors after initial habituation to a similar odor, comparable to behavioral changes seen in Kv1.3 knockout animals. In summary, we have developed novel photoactivatable Kv channels that provide new ways to interrogate neural circuits in vivo and to examine the roles of normal and disease-causing mutant Kv channels in brain function and behavior.

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Species referenced: Xenopus laevis
Genes referenced: eef1a2 rac1

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	Fig 1. Rationale and design of Opto-Kv channels. a) Structural design mapping a photoactivatable derivative of Rac1 (LOV-Rac) onto a Opto-Kv LOV-T1 fusion domain. (Left) Crystal structure of a photoactivatable Rac1 containing wild-type LOV2 (Protein Data Bank 2WKP). It shows the connection of LOV domain J-alpha helix (red) to the N-terminal beta strand of Rac1 (magenta). (Right) The LOV2 domain was mapped by homology onto the N-terminal beta strand of aKv1 T1 domain (magenta) (Protein Data Bank 1T1D). The T1 domain was tilted 45°C off axis to show the linkage similarity. A proline residue was introduced into the LOV-T1 linker to move the LOV domain into a better position to disrupt T1-T1 interactions. b) Domain structure and sequence margins for Opto-Kv channel constructs using aKv1 channel. Residue color matches that of the corresponding domain. N-terminal amino acids included in the Opto-aKv1/SL (green) and Opto-aKv1/ML (blue) variants are indicated by the colored bars. https://doi.org/10.1371/journal.pone.0248688.g001
	Fig 2. Channel expression suppressed by LOV domain and its photoactivation by light. a) Currents in Xenopus oocytes following injection of various cRNAs (aKv1/full-length and Opto-aKv1) and overnight expression at 18°C under dark or blue light conditions. Traces were elicited by voltage steps from -60 mV to +50 mV at 10 mV steps from a holding potential of -100 mV. Blue light was applied continuously for 16 hrs at 14 μW/mm2. b) Analysis of peak current amplitude at +50 mV. Results plotted as mean ± standard error (SE) (p < 0.01 Dark-Light). c) Close-up comparison of current kinetics for Opto-aKv1/FL with blue light and aKv1/FL in the dark recorded at +50 mV using elevated K+ Out. Following normalization for peak current amplitude the current waveforms are nearly identical. d) Inactivation of outward currents as measured using single exponential decay at described voltages. e) Conductance-voltage relationship of aKv1/FL channels compared to those of Opto-aKv1/FL in the dark or light. Results are shown as mean ± SEM. https://doi.org/10.1371/journal.pone.0248688.g002
	Fig 3. N-terminal variants of Opto-aKv1 show different inactivation kinetics but similar levels of induction with blue light. a) Outward currents expressed by Opto-aKv1/ML and Opto-aKv1/SL elicited by depolarizing pulses as described in Fig 2a. Application of blue light, as observed with Opto-aKv1/FL, dramatically induced surface expression of Opto-aKv1/ML and Opto-aKv1/SL. b) Overlapped traces of Opto-aKv1/FL, Opto-aKv1/ML, and Opto-aKv1/SL at +50 mV with blue light treatment. https://doi.org/10.1371/journal.pone.0248688.g003
	Fig 4. Time course of photoactivation and deactivation in oocytes. Opto-aKv1/ML channels were expressed in Xenopus oocytes overnight in the dark or with blue light exposure. Currents were elicited by +50 mV depolarization from -100 mV holding potential. a) Photoactivation of current. Oocytes incubated in the dark overnight expressed very little current; however, progressively longer exposure to blue light led to increasing current expression. b) Time course of photoactivation in an individual oocyte. Induction of current by blue light is significant within 2 hours of exposure and follows a 9 hr time constant. c) Deactivation by darkness. Oocytes exposed to blue light overnight were left in the dark and then tested at the described time points. d) Single exponential fit to dark deactivation of induced current shows a 1.5 hr time constant to return to baseline. https://doi.org/10.1371/journal.pone.0248688.g004
	Fig 5. Rapid photoinduction and deactivation of Opto-aKv1 channels from mammalian cell surface membrane. a) Photoactivation of Opto-aKv1/SL channels by blue light treatment. Transfected HEK293K cells were incubated in the dark or under blue light for 1.5 hours before voltage-clamp recordings. Currents were elicited by depolarization to +50 mV from holding at -80 mV for 400 ms. Currents were also measured after returning the cells to dark for varying amount of time until the baseline was reached. b) Current density in response to light plotted as a function of time. The time course of both photoactivation and deactivation were significantly more rapidly in HEK293K cells than in oocytes. c) Visualization of wild-type aKv1 and Opto-aKv1 expression in the dark or under blue light by immunostaining. Total Opto-aKv1 protein was obtained by permeabilizing the cells before antibody staining; surface proteins, without permeabilization. Scale bar = 20 um. d) Confirmation of increase in surface expression in response to blue light treatment. CHO-K1 cells transfected with Opto-aKv1/SL/HA were left in the dark or treated with blue light for 1.5 hours. Surface Opto-aKv1/SL channels tagged with external HA epitope (Opto-aKv1/SL/HA) were labelled with biotin before cell lysis and pulldown using streptavidin beads. Proteins separated on Western blots were probed with anti-HA antibody (1:200). https://doi.org/10.1371/journal.pone.0248688.g005
	Fig 6. Photoactivation of dominant negative Opto-aKv1/V400D subunits in mammalian cells, cerebellar granule cells, and Jurkat cells. a) Photoactivation of Opto-aKv1/V400D markedly suppresses wild-type aKv1/FL current in CHO-K1 cells. CHO-K1 cells were co-transfected with wild-type aKv1/FL and Opto-aKv1/V400D, allowed to express overnight, and were either left in the dark or exposed to blue light for 1.5 hours before recordings. Whole-cell currents were elicited by step depolarization from -90 to +80 mV from a holding potential of -80 mV. b) Current density at +80 mV under dark and blue light conditions. Data is shown as mean ± SEM. Number of experiments: n = 12 for dark and n = 7 for blue light. c) Photoactivation of Opto-aKv1/V400D heterologously expressed in cerebellar granule (CG) cells also suppresses wild-type aKv1/FL current. To isolate the aKv1/FL current, the prominent endogenous Kv4 subthreshold A-current (ISA) was inactivated by a 900 ms-long prepulse to -30 mV before the test pulse to various potentials between +80 mV and -90 mV in 10 mV increments. Light treatment was the same as in A. d) Current density at +80 mV under dark (n = 8) and blue light (n = 4). e) Suppression of endogenous Kv1.3 in Jurkat cells by photoactivation of heterologously expressed Opto-aKv1/V400D channels. f) Current density of Kv1.3 current in Jurkat cells expressing Opto-aKv1/V400D in the dark (n = 4) and blue light (n = 5) (p < 0.01). Data is shown as mean ± SEM. https://doi.org/10.1371/journal.pone.0248688.g006

	Fig 8. Expression of Opto-Kv channels in different expression systems. In the dark, Opto-Kv channel monomeric subunits are retained by the endoplasmic reticulum (ER) by trafficking machinery and shuttled for degradation because the inactive LOV domain (red) disrupts T1-dependent tetramerization and channel assembly. Upon blue light (hv) exposure, activated LOV do-main (green) frees the T1 domain to tetramerize, allowing fully assembled channels to easily traffic to the surface. (left panel) In Xenopus oocytes, blue light increases Opto-Kv current approximately 10-fold after overnight exposure at 18°C. (center panel) Blue light exposure for 1.5 hours is sufficient to rapidly increase surface expression about 3-5-fold by greatly increasing the fraction of tetrameric channels. This increase plateaus possibly due to limited subunit availability due to degradation of unassembled subunits prior to light exposure. (right panel) Neurons are making wild type channels but unassembled Opto-Kv-DN channels are shunted to degrade. Blue light treatment for 1.5 hours allows Opto-Kv-DN subunits to co-assemble with wild type dropping currents by 80%. Large effect is due to ability of one Opto-Kv-DN subunit to disrupt three wild type subunits. https://doi.org/10.1371/journal.pone.0248688.g008
	S1 Fig. Light-activated suppression of aKv1/FL current by dominant negative Opto-aKv1/V400D construct in oocytes. a) Currents by oocytes expressing aKv1/FL and Opto-aKv1/V400D incubated overnight in the dark or under blue light. Currents were elicited by 500 ms step depolarizations from -100 mV holding potential in 10 mV increments under elevated external K+ condition. b) Measurements of peak current at +50 mV under dark (n = 3) and light conditions (n = 4) (p < 0.01). Mean ± SEM. https://doi.org/10.1371/journal.pone.0248688.s001

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