Improved calcium sensor GCaMP-X overcomes the calcium channel perturbations induced by the calmodulin in GCaMP.

Molecular biology Ca V 1.3 α 1D_Long (denoted as α 1DL ) variant (NM000720, GenBank TM accession number), Ca V 2.2 (α 1B , NM_001243812.1 ), CaM and CaM 1234 were generously provided by Dr. David Yue (Johns Hopkins University). cDNA constructs of certain GCaMP variants were generous gifts from Drs. Minmin Luo and Sen Song (Tsinghua University). cDNA constructs encoding D3cpv (36323) and CaMPARI (60421) were purchased from Addgene. Inverse pericam was provided by Dr. Atsushi Miyawaki (Riken Brain Science Institute). Ca V 1.3 α 1D_Short (denoted as α 1DS ) variant was constructed by introducing a unique XbaI site following the IQ domain. YFP-tagged CaM/pcDNA3 was firstly generated by inserting YFP into pcDNA3 vector via unique KpnI and NotI sites, then CaM was fused to carboxyl termini of YFP via unique NotI and KpnI sites 22 . To target CFP and GCaMP3 to the nucleus of cells, a short NLS (PKKKRKV) was fused to both amino and carboxyl termini. NLS-CFP-NLS segment was amplified by PCR with flanking KpnI and XbaI then cloned directionally via these two unique sites into pcDNA3 expressing plasmids. NLS-GCaMP3-NLS segment was amplified by PCR with flanking BglII and NotI then cloned by replacing GCaMP3 via these two unique sites, yielding NLS-GCaMP3-NLS. IQ domain from neuromodulin ( NM_017195.3 ) with modifications to render constitutive high-affinity apoCaM binding was amplified by PCR, with the sequence encoding MGMDELYKGTAATKIQAAFRGHITRKKLKDEKKGA (denoted as CBM). The fusion of CBM-GCaMP3 (GCaMP3-X O ) was ligated with EcoRI by overlap PCR with flanking BglII and NotI then cloned into pEGFP-N1 vector. CBM-GCaMP5G (GCaMP5G-X O ) and CBM-GCaMP6m (GCaMP6m-X O ) constructs were made by replacing GCaMP3 with appropriate PCR-amplified segments via unique EcoRI and NotI sites. To cytosolic localizations, NES (LALKLAGLDIGS) was fused to carboxyl terminus of GCaMP-X O . Construct of CBM-GCaMP3-NES, CBM-GCaMP5G-NES and CBM-GCaMP6m-NES were named GCaMP3-X C , GCaMP5G-X C and GCaMP6m-X C , respectively. To specifically target the nucleus, NLS (PKKKRKV) was fused to its C-terminus of GCaMP-X O . Construct of CBM-GCaMP3-NLS was named as GCaMP3-X N . To target the membrane, Lck sequence of MGCGCSSNPEDDWMENIDVCENCHYPPPKLRIDPPDLAT was fused to the N-terminus of the sensors, then cloned into pcDNA3 vector via unique BamHI and NotI sites. The membrane-targeted versions of GCaMP3 and GCaMP3-X were denoted as GCaMP3 M and GCaMP3-X M , respectively. TagRFP was amplified by PCR with flanking KpnI and NotI and cloned directionally via these two unique sites into pcDNA3 expressing plasmids. Then GCaMP3 was amplified with flanking NotI and XbaI and cloned via these two sites into pcDNA3, yielding TagRFP-GCaMP3 construct. To make GCaM 1234 P3 construct, CaM 1234 was amplified by overlap PCR with point mutation N60D and flanking MluI and NotI then cloned by replacing CaM N60D within GCaMP3. To delete M13 and cpEGFP from GCaMP3 and GCaM 1234 P3, EGFP was amplified by PCR with flanking BglII and MluI then cloned by replacing M13 and cpEGFP via these two unique sites based on GCaMP3 and GCaM 1234 P3, respectively. For chimeric α 1DS -DCT F , α 1DS -G 12 -GCaMP3 and α 1DS -G 12 -GCaMP3ΔM13-cpEGFP, α 1DS -DCT F was adopted from our previous work 22 , 67 . G 12 segment was PCR-amplified with SpeI and XbaI sites and inserted into α 1DS as template. GCaMP3 and GCaMP3ΔM13-cpEGFP then were PCR-amplified with identical SpeI and XbaI sites and inserted into α 1DS -G 12 . Dissection and culturing of cortical neurons Cortical neurons were dissected from newborn ICR mice. Isolated tissues were digested with 0.25% trypsin for 15 min at 37 °C, followed by terminating the enzymatic reaction by DMEM supplemented with 10% FBS. The suspension of cells was sieved through a filter then centrifuged at 1000 rpm for 5 min. The cell pellet was resuspended in DMEM supplemented with 10% FBS and were plated on poly-D-lysine-coated 35 mm confocal dishes (In Vitro Scientific) or coverslips. After 4 h, neurons were maintained in Neurobasal medium supplemented with 2% B27, 1% glutaMAX-I (growth medium) for 5–6 days. Temperature should be around 37 °C with 5% CO 2 in the incubator. All animals were obtained from the laboratory animal research center, Tsinghua University. Procedures involving animals has been approved by local institutional ethical committees (IACUC, Tsinghua University).

Transfection of cDNA constructs The HEK293 cell line

(ATCC) used in this study was free of mycoplasma contamination, checked by PCR with primers 5′-GGCGAATGGGTGAGTAACACG-3′ and 5′-CGGATAACGCTTGCGACCTATG -3′ 22 . HEK293 cells were cultured in 60 mm dishes, and recombinant channels were transiently transfected according to an established calcium phosphate protocol 22 . We applied 5 μg of cDNA encoding the desired channel α 1 subunit, along with 4 μg of rat brain β 2a ( M80545 ) and 4 μg of rat brain α 2 δ (NM012919.2) subunits. Additional 2 μg of cDNA was added as required in co-transfections. All of the above cDNA constructs were driven by a cytomegalovirus promoter. To enhance expression, cDNA for simian virus 40 T antigen (1–2 μg) was also co-transfected. Cells were washed with PBS 6–8 h after transfection and maintained in culture medium of supplemented DMEM, then incubated for at least 48 h in a water-saturated 5% CO 2 incubator at 37 °C before whole-cell recordings. Neurons were cultured in 35 mm confocal dishes or coverslips, 2 μg of cDNA encoding the desired peptides were transiently transfected by Lipofectamine 2000 (Invitrogen). The opti-MEM containing plasmids and Lipofectamine 2000 was added to the Neurobasal medium for transfection. After 2 h, neurons were maintained in Neurobasal medium supplemented with 2% B27, 1% glutaMAX-I for 48 h. Infection of adeno-associated virus Virus of GCaMP6m (AAV- Syn -GCaMP6m), GCaMP6f (AAV- Syn -GCaMP6f) and GCaMP6m-X C (AAV- Syn -GCaMP6m-X C ) were provided by University of Pennsylvania Vector Core (USA) or Hanbio Biotechnology (China). Viruses were added to growth medium of the cortical neuron culture for 1–2 weeks.

Whole-cell electrophysiology

Whole-cell recordings of transfected HEK293 cells or cortical neurons were obtained at room temperature (25 °C) using an Axopatch 200B amplifier (Axon Instruments). Electrodes were pulled with borosilicate glass capillaries by a programmable puller (P-1000, Sutter Instruments) and heat-polished by a microforge (MF-830, Narishige), resulting in 1–3 MΩ resistances, before series resistance compensation of 70% or more. The internal solutions contained, (in mM): CsMeSO 3 , 135; CsCl 2 , 5; MgCl 2 , 1; MgATP, 4; HEPES, 5; and EGTA, 5; at 290 mOsm adjusted with glucose and at pH 7.3 adjusted with CsOH. The extracellular solution contained (in mM): TEA-MeSO 3 , 140; HEPES, 10; CaCl 2 or BaCl 2 , 10; 300 mOsm, adjusted with glucose and at pH 7.3 adjusted with TEAOH, all according to the previous reports 22 , 67 . Whole-cell currents were generated from a family of step depolarizations (−70 to + 50 mV from a holding potential of −70 mV) or a series of repeated step depolarizations (−10 mV from a holding potential of −70 mV). Currents were recorded at 2 kHz by the low-pass filter, and cells with Ca 2+ current amplitude less than 100 pA were not included in the analyses. Traces were acquired at a minimum repetition interval of 30 s. P/8 leak subtraction was used throughout. For patch-clamp recordings on neurons, isolated cortical neurons were cultured for 11 days in order to obtain larger calcium current. To isolate Ca V 1.3 currents, Ca V 1.2, N- and P/Q-type Ca 2+ channel currents were blocked by pre-incubating neurons in Tyrode’s solution containing 1 μM nimodipine (Sigma-Aldrich), 1 μM ω-conotoxin GVIA (Sigma-Aldrich, or Alomone Labs), 1 μM ω-conotoxin MVIIC (Sigma-Aldrich, or Alomone Labs) for 30 min before recording, according to cocktail recipes 65 , 68 , 69 , validated with our recordings in which Ca V 1.3 was the dominant component (~80%), compared to Ca V 2.3 (~16%) and Ca V 1.2 (contributing to the rest). Neurons were used within 1 h after pre-incubation and recorded in bath solution with blocker cocktail. In all electrophysiological experiments, recordings from at least four cells were analyzed ( n ≥ 4).

Immunocytochemistry

Cortical neurons which were transfected on 5th day and used on 7th day, were silenced with 1 μM TTX for 24 h to prevent AP, then transferred to 40 mM [K + ] o solution (1 μM TTX, 95 mM NaCl, 40 mM KCl, 1 mM MgCl 2 , 15 mM HEPES, 1.2 mM CaCl 2 , at 300 mOsm adjusted with glucose) or 20 mM [K + ] o solution (1 μM TTX, 115 mM NaCl, 20 mM KCl, 1 mM MgCl 2 , 15 mM HEPES, 1.2 mM CaCl 2 , at 300 mOsm adjusted with glucose) and treated for 30 min (detecting pCREB) or 5 min (detecting CaM). Then cortical neurons were rinsed briefly in phosphate-buffered saline (PBS), fixed with ice cold 4% paraformaldehyde in PBS (pH 7.4) for 20 min at the room temperature (~25 °C), then washed three times with ice-cold PBS. Fixed cells were then permeabilized with 0.3% Triton X-100 and blocked with 10% normal goat serum in PBS for 1 h at room temperature, and incubated overnight at 4 °C in primary antibodies of pCREB (Rabbit mAb #9198, Cell Signaling Technology, Species Cross-Reactivity: Human, Mouse, Rat, Dilutions: 1:1000), CREB (Rabbit mAb #9197, Cell Signaling Technology, Species Cross-Reactivity: Human, Mouse, Rat, Monkey, D. melanogaster, Dilutions: 1:500) or CaM (Rabbit mAb #5197-1, Epitomics, Species Cross-Reactivity: Human, Mouse, Rat, Dilutions: 1:500). The next day, cells were washed with PBS three times, incubated at room temperature for 2 h in 1:800 dilution of anti-rabbit-Alexa633 (Invitrogen), and washed with PBS three times (incubated with Hoechst 33342 for 5 min when nuclear counterstain was needed). Cells were imaged with ZEISS Laser Scanning Confocal Microscope (LSM710) (Carl Zeiss) and ZEN 2009 software. Fluorescence intensity was quantified and analyzed with ImageJ (NIH). Calculations of nuclear fluorescence intensity were aided with nuclear regions stained by Hoechst 33342. In all immunocytochemistry experiments, images of at least ten cells were analyzed ( n ≥ 10).

Apoptosis assay

Cortical neurons on confocal dishes were incubated with fluorescein-labelled Annexin V reagent (Keygen Biotech, 1:100 dilution) for 15 min at the room temperature (~25 °C). Cells were washed once with Tyrode’s solution and observed with a confocal laser microscope (LSM710). Fluorescence Ca 2+ imaging with GCaMP or GCaMP-X Cortical neurons were pre-incubated in 5 mM [K + ] o solution (130 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 15 mM HEPES, 2 mM CaCl 2 , at 300 mOsm adjusted with glucose) and perfused with 20 or 40 mM [K + ] o solution, then washed out by 5 mM [K + ] o . Epi-fluorescent imaging experiments were performed with an inverted fluorescence microscope (Ti-U, Nikon, Japan) and Neo sCMOS camera (Andor Technology). The light source was from the mercury lamp filtered at appropriate wavelengths for GFP by the optical filters mounted at the computer-controlled filter wheel (Sutter Instrument) for excitation, subsequently passing the dichroic mirror and the emission filters. Operations and measurements were controlled by the iQ software (Andor Technology). For confocal Ca 2+ imaging, a similar protocol was followed with ZEISS Laser Scanning Confocal Microscope (LSM710) (Carl Zeiss) and ZEN 2009 software. Fluorescence intensity ( F ) was subtracted from its background, to calculate the index of Δ F/F 0 , where F 0 is the baseline fluorescence averaged from 1 s or three or more data points at rest, and Δ F = F−F 0 . Fluorescence Ca 2+ imaging with Fura-2 Cortical neurons were cultured on coverslips and were loaded in Tyrode’s solution with 5 μM Fura-2 AM (Abcam) and 0.02% Pluronic F-127 (Invitrogen) in incubator for 30 min. Neurons were pre-incubated in 5 mM [K + ] o solution and perfused with high [K + ] o solution, then washed out by 5 mM [K + ] o . Fluorescence ratio (F 340 /F 380 ) was achieved with a standard ratiometric Ca 2+ imaging system, which included an inverted IX71epi-fluorescence microscope (Olympus), a camera of EMCCD DU-897D (Andor Technology), the light source of Lambda DG-4 (Sutter instrument), the excitation filters of 340 nm and 380 nm: FF01-340/26-25 (Semrock) and FF01-380/14-25 (Semrock), the emission filters of ~510 nm: FF01-504/12-25 (Semrock), and the dichromatic mirror: DM 400 in U-MWU2 cube (Olympus). Images were acquired and analyzed by MetaFluor software (Molecular Devices). Induction of Ca 2+ dynamics in HEK293 cells To generate Ca 2+ waveforms resembling that of single AP, the glass electrode for whole-cell patch clamp was filled with the intracellular solution containing strong Ca 2+ chelators of 10 mM BATPA. Subsequent to the standard GΩ-seal formation, 3 ms ZAP stimulation from the amplifier (Axopatch 200B) was applied for membrane break-in. Due to transient membrane rupture, a rapid Ca 2+ influx was induced (to mimic the rising phase of AP-associated Ca 2+ ). Immediately after, whole-cell configuration was stably established, and BAPTA quickly diffused into the cell, which altogether attenuated intracellular Ca 2+ (mimicking the decay phase of single-AP Ca 2+ waveform). An alternative approach to produce rapid Ca 2+ influx was by way of voltage-gated Ca V 2.2 channels overexpressed in HEK293 cells (Ca V 2.2 current amplitudes were within the range of 0.5–2 nA). A 100 ms of +10 mV voltage step was applied to depolarize the membrane and activate Ca V 2.2 channels. Under this approach, Ca 2+ influx was more controllable since Ca V 2.2 currents ( I Ca ) were simultaneously monitored by whole-cell patch clamp while GCaMP fluorescence images were acquired at 100 Hz or higher. Another simple method was also used to generate Ca 2+ dynamics by acetylcholine (Ach) activation of endogenous muscarinic receptors in HEK293 cells 5 . Bath containing 10 μM Ach (TargetMol) was briefly applied to cells and then washed out. Rise time t r was defined as the time (from the start of stimulation) to reach the half of peak Δ F/F 0 . Decay time t d was defined as the time (from the time of peak) to fall back to the half of peak Δ F/F 0 . SNR was calculated by the ratio of peak Δ F/F 0 over standard derivation (SD) of baseline signals (1 s duration). Ca 2+ imaging with outer hair cells According to previously published protocol 39 , the organ of Corti containing outer hair cells was isolated from P1 mice. For electroporation, glass electrodes (2 μM diameter) were used to deliver plasmids (1 μg/μl in 1 × HBSS) to outer hair cells. A series of three pulses was applied at 1 s intervals with a magnitude of 60 V and duration of 15 ms (ECM 830 square wave electroporator, BTX). Organ was cultured for 1–3 days in DMEM/F12 medium with 10% FBS and 1.5 μg/ml ampicillins. For Ca 2+ imaging, outer hair cells were imaged on an upright Olympus BX51WI microscope. Hair bundles of outer hair cells were stimulated with a fluid jet applied through a glass electrode filled with bath solution (1.3 mM CaCl 2 , 144 mM NaCl, 0.7 mM NaH 2 PO 4 , 5.8 mM KCl, 0.9 mM MgCl 2 , 5.6 mM glucose, and 10 mM H-HEPES, pH 7.4). Stimuli were controlled by Patchmaster 2.35 software (HEKA) and 20 psi air pressure were applied for each stimulation. Sampling rate of images was at 2 s, and duration of fluid-jet stimulation was increased from 100 to 300 ms, and then to 500 ms.

Image analysis of neurite morphology

Measurement of the length and Sholl analysis for neurites were performed with Imaris 7.7.2 (Bitplane). Only non-overlapping neurons were selected for analysis and images of at least 14 neurons were analyzed ( n ≥ 14).

Data analysis and statistics

Data were analyzed in Matlab, GraphPad Prism and OriginPro software. Standard error of the mean (S.E.M.) and Student’s t -test (two-tailed with criteria of significance: * p < 0.05; ** p < 0.01, and *** p < 0.001) were calculated when applicable, and n.s. denotes “not significant”.

Data availability

The authors declare that all data supporting the findings of this study are available within this article and the Supplementary Information File; or available from the corresponding author upon request.

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