Journal: Scientific Reports

Article Title: A rare schizophrenia risk variant of CACNA1I disrupts Ca V 3.3 channel activity

doi: 10.1038/srep34233

Figure Lengend Snippet: ( a ) Approximate locations of T797M and R1346H in extracellular loops between transmembrane helices S5 and S6 in domains II and III of hCa V 3.3. Figure highlights only S5 and S6 helices (S1–S4 missing), and pore regions of only two of four domains of a generic voltage-gated ion channel. ( b ) Amino acid sequences of Ca V 3.3 aligned for five vertebrates for regions I774 and S806 (upper), and Q1326 and V1357 (lower). Numbering according to NM_021096. The end of transmembrane helices S5 in domains II and III are marked (DIIS5, DIIIS5). Sequence alignment illustrates the high degree of conservation of amino acids in Ca V 3.3 surrounding T797 and R1346. Two putative N-glycosylation sites at asparagines 1345 and 1342 are marked (arrows; lower).

Article Snippet: T797M and R1346H were introduced on an SbfI-HindIII fragment of FLAG-tagged WT hCa V 3.3 cDNA (Origene, RC219179) and sub-cloned into pcDNA5/FRT/TO vector (Thermo Fisher Scientific) to generate full-length FLAG-tagged hCa V 3.3 cDNAs.

Techniques: Sequencing

Journal: Scientific Reports

Article Title: A rare schizophrenia risk variant of CACNA1I disrupts Ca V 3.3 channel activity

doi: 10.1038/srep34233

Figure Lengend Snippet: All membranes were cut in two; upper membranes were probed with anti-FLAG to measure hCa V 3.3 levels and lower membranes with control antibodies. ( a ) Anti-FLAG signals from Flp-In T-REx HEK293 cell lysates after induction for 24, 48, and 72 hrs with (lanes 1–3), and without (lanes 4–6), 1 μg/ml doxycycline. ( b ) Anti-FLAG signals in whole cell lysates from cells expressing WT-hCa V 3.3, untreated (1), with glycosidase (2), and same as (2) but lacking glycosidase (3). ( a , b ) Compiled figures from 4 digital images of the same gel. Protein ladder images are juxtaposed to the immunoblots; ladder lane is colored in blue. Dotted lines indicate the spliced borders of two immunoblots. ( c ) Anti-FLAG hCav3.3 levels in whole cell lysate (1–3) and biotin-surface fraction (4–6) from cells expressing WT (1, 4), T797M (T/M) (2, 5), and R1346H (R/H) (3, 6). ( d ) Anti-FLAG signal in whole cell and biotinylated (surface) preparations from cells expressing T/M and R/H shown relative to WT and normalized to controls (cadherin and β-actin). Mean ± SE values for T/M were 1.12 ± 0.08 (n = 18, whole cell) and 0.87 ± 0.13 (n = 7, surface); for R/H were 0.29 ± 0.03 (n = 18, whole cell) and 0.12 ± 0.03 (n = 7, surface). Coefficient of variation: T/M, 31% and R/H, 40% (1000 samples bootstrapping). ( e ) Anti-FLAG signals in biotinylated surface protein from cells expressing WT-hCa V 3.3, untreated (1), glycosidase exposure (2) and, conditions same as (2) but lacking glycosidase (3). ( f ) Average fraction of upper MW band relative to total hCa V 3.3 for WT: 0.80 ± 0.02 (n = 8); T/M: 0.80 ± 0.02 (n = 8); and R/H: 0.62 ± 0.05 (n = 8) from data in ( c , d ). ( g ) RT-qPCR analysis of hCa V 3.3 mRNA 72 hr after doxycycline induction expressed as fold-change relative to non-induced. Each individual point is a separate qPCR measure for 3 biological replicates and 3–4 technical replicates. Lines connect the relative levels of mRNA for three genotypes within each biological experiment. Data do not violate D’Agostino-Pearson test for normality.

Article Snippet: T797M and R1346H were introduced on an SbfI-HindIII fragment of FLAG-tagged WT hCa V 3.3 cDNA (Origene, RC219179) and sub-cloned into pcDNA5/FRT/TO vector (Thermo Fisher Scientific) to generate full-length FLAG-tagged hCa V 3.3 cDNAs.

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: A rare schizophrenia risk variant of CACNA1I disrupts Ca V 3.3 channel activity

doi: 10.1038/srep34233

Figure Lengend Snippet: ( a ) Anti-FLAG signals from total cell lysates at different time points following exposure to 0.8 μg/mL puromycin from cells expressing WT, T797M (T/M) and R1346H (R/H). All membranes were cut in two; upper membranes were probed with anti-FLAG to measure hCa V 3.3 levels and lower membranes with anti-GAPDH for normalization. ( b ) Time course of anti-FLAG hCa V 3.3 > 250kDa (closed symbols) and ~ 250kDa (open symbols) signals normalized to GAPDH and represented relative to pre-puromycin levels for WT (black), T/M (red) and R/H (blue). The >250 kDa glycosylated signals were similar among WT, T/M and R/H except at the 2 hr time point. Mean ± SE at 2hr for WT: 1.42 ± 0.10 (n = 8); T/M: 1.32 ± 0.11 (n = 5); and R/H: 0.85 ± 0.17 (n = 5), at 48 hr for WT: 0.36 ± 0.08 (n = 8); T/M: 0.27 ± 0.06 (n = 5); and R/H: 0.50 ± 0.12 (n = 5). Mean ± SE are shown for each time point. For all analysis, results shown represent at least three experimental replicates and at least two technical replicates. Data do not violate D’Agostino-Pearson test for normality, and comparisons were analyzed by one-way ANOVA with Dunnett’s post hoc test. ( c ) GAPDH levels during puromycin treatment. The level of GAPDH at each time point is normalized to GAPDH level at time 0 for each condition. Each data point represents mean ± SE for three separate cultures.

Article Snippet: T797M and R1346H were introduced on an SbfI-HindIII fragment of FLAG-tagged WT hCa V 3.3 cDNA (Origene, RC219179) and sub-cloned into pcDNA5/FRT/TO vector (Thermo Fisher Scientific) to generate full-length FLAG-tagged hCa V 3.3 cDNAs.

Techniques: Expressing

Journal: Scientific Reports

Article Title: A rare schizophrenia risk variant of CACNA1I disrupts Ca V 3.3 channel activity

doi: 10.1038/srep34233

Figure Lengend Snippet: ( a ) Left : Calcium currents recorded by whole-cell patch method from three cells expressing WT (black), T/M (red) or R/H (blue) hCa V 3.3. Currents were evoked by a series of 50 ms long test potentials from a holding potential of −100 mV. Scale bars correspond to 50 pA/pF and 10 ms. Middle : Average current-voltage plots. Plots for peak current densities for a range of test potentials (TP) with 99% confidence intervals generated by bootstrap resampling with replacement for cells expressing WT, T/M or R/H hCa V 3.3. Right : Individual current-voltage plots for average data shown in middle . ( b ) Average permeability rates ( left ) and reversal potentials ( right ) were estimated from fitting the Goldman-Hodgkin-Katz function to individual current voltage relationships shown in ( a ). Mean (circle), median (horizontal bar), interquartile range (25 th –75 th percentile, box), whiskers (range), and outliers (cross) are shown for each condition. Mean ± SE permeability rates, WT: 0.71 ± 0.08 μm/s (n = 14); T/M: 0.69 ± 0.11 μm/s (n = 12); and R/H: 0.31 ± 0.03 μm/s (n = 11). Mean ± SE reversal potentials, WT: 44.63 ± 2.02 mV (n = 14); T/M: 44.98 ± 2.86 mV (n = 12); and R/H: 38.93 ± 4.27 mV (n = 11). ( c ) Calcium currents recorded by high throughput patch method from Flp-In T-Rex HEK293 cells expressing WT, T/M or R/H hCa V 3.3. Left : Beeswarm plot of peak calcium current amplitudes for each cell line expressing hCa V 3.3 WT, T/M, and R/H. Right : cumulative frequency plot of data shown in left together with fits of each distribution. Median values calculated from parametrization of each distribution, WT: 1.30 nA; T/M: 1.30 nA; and R/H: 0.56 nA. Mean values ± SE, WT: 1.13 ± 0.08 nA (n = 128); T/M: 1.10 ± 0.06 nA (n = 126); and R/H: 0.52 ± 0.04 nA (n = 126).

Article Snippet: T797M and R1346H were introduced on an SbfI-HindIII fragment of FLAG-tagged WT hCa V 3.3 cDNA (Origene, RC219179) and sub-cloned into pcDNA5/FRT/TO vector (Thermo Fisher Scientific) to generate full-length FLAG-tagged hCa V 3.3 cDNAs.

Techniques: Expressing, Generated, Permeability, High Throughput Screening Assay

Journal: Scientific Reports

Article Title: A rare schizophrenia risk variant of CACNA1I disrupts Ca V 3.3 channel activity

doi: 10.1038/srep34233

Figure Lengend Snippet: Recordings are from cell-attached patches from tsA201 cells transiently expressing WT, T/M and R/H hCa V 3.3. ( a ) Single WT hCa V 3.3 channel currents evoked by step depolarizations from −80 mV to −20 mV, upper panel . Ensemble current trace generated by adding multiple single channel traces recorded at −20 mV, lower panel ; ( b ) Single hCa V 3.3 channel tail currents resolved immediately on membrane hyperpolarization to −50 mV from a depolarizing step to +60 mV (used to open the channels), upper panel . Tail currents are relatively large because of the large driving force at negative membrane potentials—although they close rapidly. Lower panel shows an ensemble tail current reconstructed from adding multiple single channel tail currents. Closed state is labeled (dotted line). ( c ) Average single Ca V 3.3 channel current amplitudes at different test potentials (TP, left panel ) for each clone. Single channel conductances were estimated from slopes of single channel current (i)-voltage relationships ( right panel ). Mean ± SE, WT: 14.0 ± 0.8 pS (n = 8); T/M: 13.3 ± 0.27 pS (n = 3); and R/H: 13.7 ± 0.6 pS (n = 5). In each case, N corresponds to the number of individual patch recordings but each dataset represents measurements of >100 individual channel openings. Averages are shown with 99% confidence intervals calculated using bootstrap with resampling.

Article Snippet: T797M and R1346H were introduced on an SbfI-HindIII fragment of FLAG-tagged WT hCa V 3.3 cDNA (Origene, RC219179) and sub-cloned into pcDNA5/FRT/TO vector (Thermo Fisher Scientific) to generate full-length FLAG-tagged hCa V 3.3 cDNAs.

Techniques: Expressing, Generated, Labeling

Journal: Scientific Reports

Article Title: A rare schizophrenia risk variant of CACNA1I disrupts Ca V 3.3 channel activity

doi: 10.1038/srep34233

Figure Lengend Snippet: ( a ) Left : hCa V 3.3 tail currents from cells expressing WT, T/M, and R/H clones. Currents were recorded after membrane potential is hyperpolarized to −80 mV from a series of steps (−80 mV to +60 mV). Current amplitudes were normalized. ( b ) Left : I/I max activation at −80 mV from different test potentials. Averages are shown with 99% confidence intervals generated by bootstrap analysis for the three cell lines and; right : individual activation curves for each recording. The activation curve is distorted at stronger depolarizations that induce inactivation during the test pulse, but there is no difference among the three clones. ( c ) Left : Average V 1/2 values estimated from fitting two Boltzmann functions to individual activation curves in b . Average (circle), median (horizontal bar), interquartile range (25 th –75 th percentile, box), whiskers (range), and outliers (cross) for each condition. Mean values ± SE for V 1/2-negative , WT: −40.0 ± 0.98 mV; T/M: −38.8 ± 0.35 mV; and R/H: −37.6 ± 0.99 mV; for V 1/2-positive , WT: 19.9 ± 2.2 mV; T/M: 22.6 ± 1.46 mV; and R/H: 15.4 ± 2.54 mV; for k , WT: 13.4 ± 0.6 mV; T/M: 14.7 ± 0.8 mV; and R/H: 14.3 ± 0.8 mV. ( d ) Calcium currents from cells expressing different hCa V 3.3 as described above for panels a and b . Left: Currents activated by depolarizations to 0 mV, −20 mV, and −40 mV. Current amplitudes were normalized for visual comparison. ( e ) Time constants estimated from fitting the rising phase of calcium currents evoked by different test potentials (TP) are averaged and plotted as described above for panel b . ( f ) Closing rate (τ closing ) at −60 mV for WT, T/M and R/H hCa V 3.3 channels. −60 mV was chosen to minimize influence from the differences in current size between R/H and WT. Data are shown similar to panel b . Mean values ± SE, τ closing for WT: 5.33 ± 0.75 ms; T/M: 5.38 ± 0.88 ms; and R/H: 4.62 ± 0.46 ms. n values ( b – f) , WT: n = 8; T/M: n = 8; and R/H: n = 8.

Article Snippet: T797M and R1346H were introduced on an SbfI-HindIII fragment of FLAG-tagged WT hCa V 3.3 cDNA (Origene, RC219179) and sub-cloned into pcDNA5/FRT/TO vector (Thermo Fisher Scientific) to generate full-length FLAG-tagged hCa V 3.3 cDNAs.

Techniques: Expressing, Clone Assay, Activation Assay, Generated

Journal: Scientific Reports

Article Title: A rare schizophrenia risk variant of CACNA1I disrupts Ca V 3.3 channel activity

doi: 10.1038/srep34233

Figure Lengend Snippet: P values calculated using the 2-sample Kolmogorov-Smirnov test ( a ) Left : Representative traces to determine the availability of channels to open upon depolarization (voltage-dependence of inactivation). Voltage-dependent inactivation was obtained using a pre-pulse protocol. 2 s inactivating pre-pulses were applied from −110 mV to −10 mV in 10 mV steps; each pre-pulse was followed with a test pulse to −20 mV. Middle : voltage dependence of inactivation for WT, T/M and R/H hCav3.3 currents. Symbols represent mean and shaded areas correspond to 95% bootstrapped confidence interval. Right : Individual voltage dependent inactivation curves from each genotype are also shown. ( b ) Inactivation curves were fitted to a Boltzmann function. V 1/2 and slope factor ( k) were similar among the three genotypes. Average (circle), median (horizontal bar), interquartile range (25 th –75 th percentile, box), whiskers (range), and outliers (cross) are shown for V 1/2 and k. Left: V 1/2 mean ± SE, WT: −67.1 ± 0.89 mV (n = 11); T/M: −65.3 ± 1.30 mV (n = 6); and R/H: −69.4 ± 1.4 mV (n = 5). Right : k mean ± SE, WT: 6.90 ± 0.21 mV (n = 11); T/M: 6.45 ± 0.37 mV (n = 6); and R/H: 6.93 ± 0.48 mV (n = 5). ( c ) Left : representative traces depicting the rate of decay of the calcium current (open-state inactivation) during the test pulse for WT, T/M, R/H hCa V 3.3 channels. Traces were normalized to enable comparisons. Middle : the decaying phase of calcium currents at several voltages was fitted to a single exponential. The rate of decay increased with test potential depolarization, and the time constants were similar among the three genotypes at all voltages analyzed. Right : time constants of inactivation for individual cells.

Article Snippet: T797M and R1346H were introduced on an SbfI-HindIII fragment of FLAG-tagged WT hCa V 3.3 cDNA (Origene, RC219179) and sub-cloned into pcDNA5/FRT/TO vector (Thermo Fisher Scientific) to generate full-length FLAG-tagged hCa V 3.3 cDNAs.

Techniques:

Journal: Sensors (Basel, Switzerland)

Article Title: Multi-Serial Adaptive Bus Interface with Integrated Monitoring and Plug-And-Play Connectivity

doi: 10.3390/s25247638

Figure Lengend Snippet: CAN transmission of 1-byte data packet containing the ASCII character “W” using the SN65HVD230 transceiver at 1 Mbps ( top ). Bit-level analysis of the CAN2.0A frame ( bottom ).

Article Snippet: In this test, a 3.3 V SN65HVD230 CAN transceiver from Texas Instruments (Dallas, TX, USA) was connected to an experimental STM32F4-Discovery development board [ ], configured for CAN 2.0A at the maximum data rate of 1 Mbps.

Techniques: Transmission Assay