A genetically encoded actuator boosts L-type calcium channel function in diverse physiological settings

Abstract

L-type Ca²⁺ channels (Ca_V1.2/1.3) convey influx of calcium ions that orchestrate a bevy of biological responses including muscle contraction, neuronal function, and gene transcription. Deficits in Ca_V1 function play a vital role in cardiac and neurodevelopmental disorders. Here, we develop a genetically encoded enhancer of Ca_V1.2/1.3 channels (GeeC_L) to manipulate Ca²⁺ entry in distinct physiological settings. We functionalized a nanobody that targets the Ca_V complex by attaching a minimal effector domain from an endogenous Ca_V modulator-leucine-rich repeat containing protein 10 (Lrrc10). In cardiomyocytes, GeeC_L selectively increased L-type current amplitude. In neurons in vitro and in vivo, GeeC_L augmented excitation-transcription (E-T) coupling. In all, GeeC_L represents a powerful strategy to boost Ca_V1.2/1.3 function and lays the groundwork to illuminate insights on neuronal and cardiac physiology and disease.

Fig. 1.. Leveraging Lrrc10 modulation of Ca_V1.2 to develop a genetically encoded enhancer.

(A) Flow cytometric FRET 2-hybrid assay shows robust Ca_V1.2 binding to Lrrc10. [K_a,EFF = 8.3 × 10⁻⁵ ± 4.8 × 10⁻⁵ arbitrary fluorescence units (a.f.u.), n = 8]. Left: Schematic. Right: Binned data of FRET efficiency (E_D) versus free acceptor concentration (A_free). Each dot represents means ± SEM. (B) Exemplar traces show Ca_V1.2 without (left) or with Lrrc10 (right) evoked by 15-ms test pulses from −50 to +80 mV in 10-mV intervals. (C) Lrrc10 up-regulates peak Ca_V1.2 current density (J_peak). Each dot represents mean ± SEM. n = 8 (baseline) and 15 cells (+Lrrc10) from three transfections. (D) FRET analysis confirms interaction of Na_V1.5 with Lrrc10. (K_a,EFF = 1.4 × 10⁻⁴ ± 1.8 × 10⁻⁵, n = 4). (E) Exemplar multichannel recordings of Na_V1.5 with (right) or without Lrrc10 (left). (F) Bar graph summary of I_NaL measured as R_persist. Each bar, means ± SEM. n = 19 (no Lrrc) and 13 cells (+Lrrc10) from three transfections. (G) FRET analysis shows differential binding of Lrrc10 NT and CT with Ca_V1.2. Format as in (A). (H) Bar graph summary of K_a,EFF for Ca_V1.2 association Lrrc10 fragments. ****P < 0.0001 by one-way analysis of variance (ANOVA) followed by Tukey’s test. (I) Both Lrrc10 CT (left) and NT (right) fail to up-regulate Ca_V1.2. (J) Population J_peak-V relationship confirms minimal changes in Ca_V1.2 with Lrrc10 NT or CT. n = 9 (CT) and 27 cells (NT) from three transfections. (K) Schematic shows AlphaFold3-predicted structure of Lrrc10 and design of GeeC_L. (L) GeeC_L (right) but not nbF3 (left) enhances Ca_V1.2. (M) Population data confirms a robust increase in J_Peak of Ca_V1.2 with GeeC_L compared to Nb.F3 alone. n = 14 cells each from three transfections. (N) Exemplar multichannel recordings of Na_V1.5 show low I_NaL with GeeC_L. (O) Bar graph shows minimal increase in I_NaL with GeeC_L. Blue bar, means ± SEM. Black, Na_V1.5; red, Lrrc10 from (F).

Fig. 2.. GeeC_L selectively enhances Ca_V1.2/1.3 currents.

(A) Coexpression of GeeC_L (red) increases Ca_V1.3 currents in HEK 293 cells compared to nb.F3 (blue). Inset shows exemplar recordings in the presence of nb.F3 or GeeC_L. Error bars show mean and SEM. (B) GeeC_L minimally perturbs peak current density of Ca_V1.4 channels. Format as in (A). (C to E) GeeC_L fails to alter peak current density of Ca_V2.1 (C), Ca_V2.2 (D), and Ca_V2.3 (E) channels. Format as in (A). (F) Bar graphs compare peak current density for various Ca_V1/2 channels in the presence of nb.F3 (blue) or GeeC_L (red). Error bars show mean and SEM. Statistical analysis: Kolmogorov-Smirnov normality test followed by Mann-Whitney test (Ca_V1.2, Ca_V1.3, Ca_V2.1) and unpaired Student’s t test (Ca_V1.3 Ca_V2.2, and Ca_V2.3). *P < 0.05 and ***P < 0.001. n = 14 and 24 (−/+ GeeC_L, Ca_V1.2); n = 7 and 9 (−/+ GeeC_L, Ca_V1.3); n = 6 and 9 (−/+ GeeC_L, Ca_V1.4); n = 10 and 11 (−/+ GeeC_L, Ca_V2.1); n = 9 and 7 (−/+ GeeC_L, Ca_V2.2); and n = 13 and 10 (−/+ GeeC_L, Ca_V2.3) from two to three transfections.

Fig. 3.. GeeC_L up-regulates Ca_V1.2 function by enhancing channel openings.

(A) Exemplar cell–attached single-channel recordings of Ca_V1.2 reconstituted in HEK 293 cells. Slanted gray curve represents unitary conductance. Each downward deflection indicates channel openings. (B and C) Coexpression of GeeC_L increases channel openings, while nb.F3 yields minimal change. Format as in (A). (D) Ensemble average P_O-voltage relationship for Ca_V1.2 shows that GeeC_L increases peak P_O compared to control conditions or in the presence of nb.F3. n = 6 (control), n = 6 (nb.F3), and n = 7 (GeeC_L) from three independent transfections. (E) Schematic shows dual labeling approach to quantify surface expression of Ca_V1.2. The pore-forming α_1C subunit is engineered to contain a BTX binding site in DII S5-S6 loop and a YFP at the C terminus. Surface expression can be quantified by labeling with BTX conjugated to Alexa Fluor 647 (S_A647), while total expression is determined by measuring YFP fluorescence (S_YFP). (F) Flow cytometric analysis shows baseline levels of Ca_V1.2 surface membrane trafficking in the presence of β_2b subunits. (G and H) Coexpression of GeeC_L (G) or nb.F3 (H) minimally perturbs surface expression of Ca_V1.2. (I) Population data confirms minimal change in surface-membrane expression of Ca_V1.2 in the presence of either GeeC_L (red) or nb.F3 (blue), normalized to control. Each dot represents geometric mean of Alexa Fluor 647 staining from an individual experiment. Bars show mean and SEM. from n = 4 transfections.

Fig. 4.. GeeC_L enhances endogenous Ca_V1.2 in mouse-derived cardiomyocytes.

(A) Brightfield (top) and epifluorescence (bottom) images show a cardiomyocyte expressing GeeC_L, with mCherry bicistronically expressed. Scale bars, 10 μm. (B) Top: Exemplar traces of endogenous L-type currents in 2-day cultured cardiomyocytes evoked in response to 300-ms voltage steps to various potentials. Bottom: Population data show baseline peak current density. Error bars represent mean and SEM. n = 11 cells from three mice. (C) Adenoviral expression of GFP minimally perturbs L-type current density. n = 8 cells from three mice. Format as in (B). (D) GeeC_L markedly up-regulates endogenous L-type channels. n = 11 cells from three mice. Format as in (B). (E) Nb.F3 by itself failed to appreciably perturb peak current density. Format as in (B). n = 8 cells from two mice. eGFP, enhanced GFP.

Fig. 5.. GeeC_L boosts E-T coupling in neurons.

(A) Confocal images show increased nuclear accumulation of pCREB (Alexa Fluor 647) following cell depolarization with extracellular solution containing 40 mM K⁺. White dashed circles mark the nuclear region for fluorescence quantification based on 4′,6-diamidino-2-phenylindole (DAPI) staining. Scale bars, 5 μm. (B) Expression of GFP has minimal effect on nuclear pCREB levels following depolarization. Scale bars, 5 μm. (C) Expression of GeeC_L increases nuclear pCREB staining following depolarization. Scale bars, 5 μm. (D) Population data compares nuclear pCREB signal following depolarization. Bars show means ± SEM. Statistical analysis: Kruskal-Wallis test followed by Dunn’s multiple comparisons test. ****P < 0.0001. n = 133 (control, resting), n = 122 (control; with depolarization), n = 95 (GFP), and n = 110 (GeeC_L) cells from six independent cultures. (E) To probe functionality of GeeC_L in vivo, AAV9 encoding either GFP or GeeC_L/mCherry was stereotactically injected into the mouse hippocampus. Confocal image of a coronal slice of mouse hippocampi at ×10 magnification shows robust expression of GeeC_L (mCherry) or GFP control AAV9 (top) and pCREB signal (bottom). pCREB staining is qualitatively higher in the right hemisphere with GeeC expression. Scale bars, 500 μm. (F) Higher magnification (×40) confocal images of mouse dorsal hippocampal neurons showing GFP (left) or mCherry (right) and corresponding pCREB staining. Scale bars, 20 μm. (G) Representative coronal slice of medial prefrontal cortex at ×10 magnification shows either GeeC_L or GFP expression (left) and corresponding pCREB staining (right). Scale bars, 200 μm. (H) Higher magnification images of mice prefrontal cortex neurons. Top: GFP or GeeC_L expression. Bottom: pCREB. Scale bars, 20 μm. (I) Population data confirms enhanced pCREB staining in GeeC_L-expressing prefrontal cortex neurons when compared to GFP-infected controls. Format as in (D). Statistical analysis: Mann-Whitney test. ****P < 0.0001.

Fig. 6.. GeeC_L boosts E-T coupling in RTT neurons.

(A) Exemplar confocal images show robust increase in nuclear pCREB staining following mild membrane depolarization in wild-type hESC-derived neurons. Left: Baseline. Right: Following 40 mM K⁺ stimulation. Scale bars, 5 μm. (B) Expression of GFP in wild-type neurons minimally perturbs recruitment of nuclear pCREB. Scale bars, 5 μm. (C) Accumulation of pCREB in the nucleus following membrane depolarization is markedly reduced in RTT-like neurons. Scale bars, 5 μm. (D) L-type channel up-regulation by GeeC_L enhances pCREB signaling in RTT neurons. Scale bars, 5 μm. (E) Bar graph summarizes population data showing increase in pCREB following membrane depolarization in both wild-type and RTT neurons. For statistical analysis, Kruskal-Wallis test followed by Dunn’s multiple comparisons test. ****P < 0.0001. n = 73 (uninfected, wild type), n = 63 (GFP, wild type), n = 128 (GFP, RTT), and n = 96 (GeeC_L, RTT) cells from three independent cultures. (F to H) Confocal images show differences in overall morphology of wild-type (F) versus RTT neurons in the presence of GFP (G) or GeeC_L (H). Blue, DAPI. Yellow: Tuj1. Red, mCherry. Green, GFP. Scale bars, 10 μm. (I) Bar graph quantifies changes in soma size in both wild-type and RTT neurons. Each bar and error represents means ± SEM. For statistical analysis, Kruskal-Wallis test followed by Dunn’s multiple comparisons test. *P = 0.0114 and ****P < 0.0001. n = 44 (uninfected, wild type), n = 20 (GFP, wild type), n = 155 (GFP, RTT), and n = 120 (GeeC_L, RTT) cells from three independent cultures.