Optogenetic Control of Voltage‐Gated Calcium Channels

Abstract

Voltage‐gated Ca²⁺ (Ca_V) channels mediate Ca²⁺ entry into excitable cells to regulate a myriad of cellular events following membrane depolarization. We report the engineering of RGK GTPases, a class of genetically encoded Ca_V channel modulators, to enable photo‐tunable modulation of Ca_V channel activity in excitable mammalian cells. This optogenetic tool (designated optoRGK) tailored for Ca_V channels could find broad applications in interrogating a wide range of Ca_V‐mediated physiological processes.

Voltage‐gated Ca²⁺ (Ca_V) channels constitute the major route of Ca²⁺ entry into cells of the nervous and cardiovascular systems, as well as other electrically excitable cells.1 Ca_V channels respond to membrane depolarization to permit Ca²⁺ influx, thereby playing instrumental roles in Ca²⁺‐dependent physiological processes, including neurotransmitter/hormone release, gene expression, and muscle contraction.1b, 1c Deregulated Ca_V channels can give rise to pathophysiological conditions ranging from cardiovascular disorders to psychiatric conditions.2 Consequently, Ca_V channels are important targets for therapeutic intervention and physiological regulation.3 Currently widely used Ca_V channel blockers (e.g., dihydropyridines) have prominent drawbacks, including off‐target toxicity, lack of spatial control, and non‐reversibility.3b, 4 New interventional approaches to control Ca_V channels are thus needed.

Optogenetics, which involves the incorporation of synthetic photosensitive modules into cells of living tissues to control cellular activities with high spatiotemporal precision, provides an ideal solution to overcome the drawbacks associated with conventional Ca_V channel blockers.5 The Ras‐like GTPases Rad/Rem/Gem/Kir (RGK), which function as negative regulators of Ca_V channels, are considered to be the prime candidates for generating optogenetic tools to modulate Ca_V channels.6 Given that membrane anchoring is necessary for RGKs to exert their suppressive effects on Ca_V channels,6b, 7 we reasoned that Ca_V channels could be remotely modulated by harnessing light to control the translocation of RGK to the plasma membrane (PM). We therefore engineered a set of optogenetic constructs by using a light‐sensitive heterodimerization system to control the subcellular localization of engineered Rem (Scheme 1 and Figure S1 in the Supporting Information). We chose the optical dimerizer pair iLID (LOV2‐ssrA) and sspB because of their small size, fast photoresponsive kinetics, low background interaction, and minimized perturbation to endogenous signaling pathways.8

Scheme 1.

Design of optoRGK to photo‐tune CaV channel activity. Spatiotemporal control of the Rem1 core domain is achieved by utilizing the LOV2‐ssrA/sspB optical dimerizer pair. The light‐inducible cytosol‐to‐PM translocation of Rem enables inducible suppression of Ca_V channel activity. Green crescent=1^st photodimerizer component (sspB), blue structure=membrane tethered LOV2, pink sphere=2^nd photodimerizer component (ssrA), LOV2+ssrA=improved light‐induced dimer (iLID) protein.

To enable light‐inducible cytosol‐to‐PM translocation of engineered Rem, we set out to install sspB into different positions of Rem_1‐266 tagged with the red fluorescent protein mCherry (mCherry‐Rem_1‐266) via flexible linkers with varying lengths (Figures S1–3). In parallel, we tethered iLID tagged with the yellow fluorescent protein Venus (Venus‐iLID) to the PM with a C‐terminal PM‐targeting sequence (CAAX) derived from KRas4B8 or the PM‐tethering motif (Lyn11) from the tyrosine‐protein kinase Lyn.9 We then co‐transfected these two constructs into HeLa cells and examined the reversible recruitment of cytosolic Rem_1‐266 to the PM (Figure 1 a, Figures S1–4). In the dark, mCherry‐Rem_1‐266–sspB was evenly distributed in the cytosol (Figure 1 a). Upon blue light illumination, photoexcitation produced a covalent adduct between residue C450 of LOV2 and the cofactor FMN, thereby exposing the caged ssrA component of iLID to restore its interaction with sspB. Consequently, mCherry‐Rem_1‐266–sspB was recruited toward the PM within seconds (t_1/2, _on=3.2±1.0 sec). Upon withdrawal of light, mCherry‐Rem_1‐266–sspB dissociated from the PM‐resident ssrA and diffused back to the cytosol (t_1/2, _off=23.0±2.4 sec; Figure 1, Figure S3, and Movie S1). After screening over 20 constructs with different combinations of key elements (various Rem fragments, linkers, PM‐targeting motifs, and insertion sites), we identified mCherry‐Rem1_1‐266–sspB/iLID‐CAAX as an ideal candidate because it exhibited an optimal performance with high sensitivity and relatively fast kinetics (Figures S1–S3). To enable a near 1:1 co‐expression of the two components using a single construct within the same cells, we used a multicistronic expression system by utilizing the self‐cleaving 2A peptide (P2A)to generate fluorescent protein (FP)‐tagged (mCherry or CFP) Rem1_1‐266–sspB‐P2A‐iLID‐CAAX.10 We named the system optoRGK and used the FP‐tagged constructs for further characterization and applications in excitable cells.

Figure 1.

Visualization of the reversible recruitment of the Rem core domain (Rem_1‐266, without the C‐terminus) to the PM in response to blue‐light illumination. a) Representative confocal images showing light‐inducible cytosol‐to‐PM translocation of mCherry‐Rem1_1‐266–sspB in HeLa cells co‐expressing Venus‐iLID‐CAAX. The images represent the same cell in the dark (black bar) or exposed to blue light at 470 nm (40 μW mm⁻²; blue bar). Scale bar: 5 μm. b) Quantification of cytosolic mCherry signals of optoRGK over five repeated light–dark cycles. n=34 cells from three independent experiments. Error bars denote the SEM.

To determine whether optoRGK could photo‐modulate Ca_V channels, we expressed optoRGK in HEK293 cells stably expressing the human Ca_V1.2 channel components (HEK‐Ca_V1.2)11 and evaluated membrane‐depolarization‐induced Ca²⁺ entry with a red genetically encoded Ca²⁺ sensor jRGCaMP1b12 (Figure 2 a,b) or the green Ca²⁺ dye Fluo‐4 (Figure S3). 50 mm potassium chloride (KCl) was employed to induce membrane depolarization. In the dark, addition of KCl elicited a pulse of Ca_V‐mediated Ca²⁺ influx in both control and optoRGK‐expressing cells (Figure 2, 1^st cycle). Upon blue‐light illumination, cells overexpressing optoRGK showed a significant reduction in KCl‐induced Ca²⁺ entry compared to control cells (Figure 2 a, 2^nd cycle). Furthermore, Ca²⁺ influx could be restored in the absence of blue light (Figure 2 a, 3^rd cycle), thereby attesting to the reversibility of optoRGK in the regulation of Ca_V channels. In parallel, we performed electrophysiological studies to independently confirm optoRGK‐mediated, light‐switchable modulation of Ca_V channels in HEK293‐Ca_V1.2 cells. In the dark, cells expressing optoRGK showed robust whole‐cell currents with a typical Ca_V I‐V relationship and amplitudes similar to those of control cells (maximal peak current density≈21.2 pA/pF, n=12; Figure 2 c,d). However, upon receiving blue‐light stimulation, the amplitudes of depolarization‐induced whole‐cell currents were significantly diminished (maximal peak current density≈6.3 pA/pF, n=15; Figure 2 c,d). These results clearly established optoRGK as a genetically encoded light‐switchable channel modulator that allows optical inhibition of Ca_V channels in excitable cells.

Figure 2.

OptoRGK‐mediated photoswitchable inhibition of Ca²⁺ entry through Ca_V1.2 channels. a) Ca²⁺ influx in HEK‐Ca_V1.2 cells transiently expressing optoRGK and the red Ca²⁺ sensor jRCaMP1b with and without blue‐light stimulation. Cells transfected with the empty vector are used as control. Membrane‐depolarization‐induced Ca²⁺ entry was elicited by adding 50 mm KCl (black line below the curves; three repeated cycles) to transfected cells. Blue line represents light stimulation under 470 nm with a power density of 40 μW mm⁻². b) Bar graphs showing the statistical results of mean Ca²⁺ entry for each cycle. c) The current–voltage relationships of Ca_V channels in HEK‐Cav1.2 cells transfected with optoRGK. Cells were either shielded from light or exposed to blue light prior to electrophysiological recording. d) Bar graphs showing the statistical results of peak whole‐cell currents induced by pulses of +10 mV depolarization (pA/pF) in HEK‐Ca_V1.2 cells before and after photo‐stimulation. All data were presented as mean±SEM. **P<0.01 (paired Student's t‐test).

We next moved on to test optoRGK in C2C12 cells, a mouse myoblast cell line13 with functional Ca_V channels.6b Again, we observed light‐dependent inhibition of Ca²⁺ influx in this excitable cell line (Figure S5). To further test optoRGK in more physiologically relevant systems, we introduced it into HL‐1 cardiomyocytes, a well‐characterized atrial myocyte culture line derived from the adult mouse that retains many of the differentiated properties of cardiac cells,14 including rhythmic oscillations of cytosolic Ca²⁺ (Movie S2). By using Fluo‐4 as the Ca²⁺ indicator, we first evaluated rhythmic Ca²⁺ oscillations in HL‐1 cells with and without overexpression of full length Rem or its truncated version Rem_1‐266 (Figure S6, Movies S3 and S4). Both control cells and HL‐1 cells transfected with mCherry‐Rem_1‐266–sspB (Figure S6b) exhibited rhythmic Ca²⁺ oscillations, while cardiomyocytes expressing the full‐length Rem failed to evoke Ca²⁺ transients (Figure S6a). These findings are consistent with the results obtained from C2C12 myoblast cells (Figure S5) and other types of excitable cells.15

Having validated the use of HL‐1 cardiomyocytes to test our tool, we next examined the rhythmic oscillations of cytosolic Ca²⁺ (Fluo‐4 signals as readout) in HL‐1 cells expressing optoRGK. Upon blue‐light illumination, mCherry‐Rem1_1‐266–sspB translocated from the cytosol to close to the PM within several seconds (Figure S7), accompanied by the attenuation of oscillatory Ca²⁺ signals (Figure 3 a, bottom, and Movie S5). By contrast, the control cells displayed regular Ca²⁺ oscillations under blue light. To further validate whether such action was reversible in HL‐1 cells, we used the red Ca²⁺ indicator Cal‐590 (excitation at 562 nm without pre‐activating optoRGK) rather than Fluo‐4 to monitor Ca²⁺ oscillations (Figure 3 b). HL‐1 cells expressing optoRGK showed regular Ca²⁺ spikes in the dark. However, upon blue‐light illumination, the rhythmic oscillations were substantially attenuated. Notably, regular Ca²⁺ oscillations were restored in the same HL‐1 cardiomyocyte after removal of the light source (Figure 3 b).

Figure 3.

OptoRGK‐mediated light‐inducible inhibition of the rhythmic oscillations of cytosolic Ca²⁺ in cardiac cells. a) Ca²⁺ oscillations reported by Fluo‐4 in HL‐1 cells with (bottom) and without (top) expression of optoRGK. Kymographs of Fluo‐4 signals in a representative HL‐1 cell are shown above the traces. Excitation was set at 488 nm to record Fluo‐4 signals while simultaneously photoactivating optoRGK. b) Ca²⁺ oscillations in HL‐1 cells monitored by Cal‐590. Blue bar=blue light illumination at 470 nm (40 μW mm⁻²).

Taken together, compared with traditional small‐molecule Ca_V channel blockers that often lack reversibility, selectivity, and tissue‐specificity, engineered RGK proteins could serve as promising candidates to enable spatiotemporal control of Ca_V channels with a simple pulse of light. This study complements the recent development of engineered stromal interaction molecule 1 (STIM1) to photo‐regulate endogenous Ca²⁺ channels in mammalian cells (e.g., optoSTIM116 and Opto‐CRAC17). We anticipate that the optoRGK tool developed in the current study will find broad applications in interrogating a wide range of Ca²⁺‐dependent physiological processes in mammals.

Proof‐of‐concept experiments have already demonstrated the potential of using RGK to treat heart disease.18 To test potential in vivo applications, we plan to express optoRGK in the atrioventricular node of rodent models with atrial fibrillation disease,19 and examine whether photostimulation could suppress aberrant atrioventricular nodal conduction to intervene atrial fibrillation.

Given that RGK proteins primarily exert suppressive effects on high‐voltage‐activated Ca²⁺ (Ca_V1/Ca_V2) channels20 and that LOV2‐based photoswitches have relatively slow kinetics, the efficacy of optoRGK will likely depend on the distribution and endogenous levels of Ca_V channel subtypes in different cell types and tissues. In its current configuration, optoRGK is well suited to modulate cardiomyocytes because of the abundant expression of l‐type Ca_V1.1/Ca_V1.2 channels and the relatively long duration of cardiac action potentials. Its compatibility with neurons and other types of electrically excitable cells (e.g., pancreatic beta cells) remains to be tested.

Conflict of interest

The authors declare no conflict of interest.

Supporting information

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G. Ma, J. Liu, Y. Ke, X. Liu, M. Li, F. Wang, G. Han, Y. Huang, Y. Wang, Y. Zhou, Angew. Chem. Int. Ed. 2018, 57, 7019.