A rare schizophrenia risk variant of CACNA1I disrupts CaV3.3 channel activity

Figures

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  Figure 1. hCaV3.3 variants, T797M and R1346H, are predicted to be extracellular. (a) Approximate locations of T797M and R1346H in extracellular loops between transmembrane helices S5 and S6 in domains II and III of hCaV3.3. 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 CaV3.3 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 CaV3.3 surrounding T797 and R1346. Two putative N-glycosylation sites at asparagines 1345 and 1342 are marked (arrows; lower).

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  Figure 2. R1346H affects surface expression of hCaV3.3. All membranes were cut in two; upper membranes were probed with anti-FLAG to measure hCaV3.3 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-hCaV3.3, 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…

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  Figure 3. Glycosylated and non-glycosylated hCaV3.3 signals have different decay time courses after inhibition of protein translation. (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 hCaV3.3 levels and lower membranes with anti-GAPDH for normalization. (b) Time course of anti-FLAG hCaV3.3 > 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…

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  Figure 4. Calcium currents in Flp-In T-Rex HEK293 cells expressing R1346H (R/H) variant are smaller compared to those in cells expressing WT and T797M (T/M). (a) Left: Calcium currents recorded by whole-cell patch method from three cells expressing WT (black), T/M (red) or R/H (blue) hCaV3.3. 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 hCaV3.3. 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 (25th–75th 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…

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  Figure 5. Single hCaV3.3 channel currents are unaffected by T797M (T/M) and R1346H (R/H). Recordings are from cell-attached patches from tsA201 cells transiently expressing WT, T/M and R/H hCaV3.3. (a) Single WT hCaV3.3 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 hCaV3.3 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 CaV3.3 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…

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  Figure 6. Activation and deactivation properties of CaV3.3 currents in cells expressing WT, T797M (T/M), and R1346H (R/H) hCaV3.3 are similar. (a) Left: hCaV3.3 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/Imax 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 V1/2 values estimated from fitting two Boltzmann functions to individual activation curves in b. Average (circle), median (horizontal bar), interquartile range (25th–75th percentile, box), whiskers (range), and outliers (cross) for each condition. Mean values ± SE for V1/2-negative, WT: −40.0 ± 0.98 mV; T/M: −38.8 ± 0.35 mV; and R/H: −37.6 ± 0.99 mV; for V1/2-positive, WT: 19.9 ± 2.2 mV; T/M: 22.6 ± 1.46 mV; and…

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  Figure 7. Inactivation properties were similar among WT, T797M (T/M) and R1346H (R/H) hCaV3.3 channels. 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. V1/2 and slope factor (k) were similar among the three genotypes. Average (circle), median (horizontal bar), interquartile range (25th–75th percentile, box), whiskers (range), and outliers (cross) are shown for V1/2 and k. Left: V1/2 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…

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  Figure 8. hCaV3.3 R1346H (R/H) variant impairs rebound burst firing in TRN neuron simulations. (a) Simulations of membrane voltage in model TRN neurons responding to hyperpolarizing or depolarizing current injections. Left: WT neuron fires a burst of rebound action potentials following 200 ms negative, hyperpolarizing current injections of different magnitudes (upper) and bursts of action potentials during the depolarizing current injection (lower). Right: Neuron with 72.5% of WT CaV3.3 current density to approximate heterozygosity (50% R/H and 50% WT) does not exhibit rebound bursting following hyperpolarization. (b) Relationship between number of rebound spikes and size of hyperpolarizing current injection for a range of CaV3.3 current densities. Percent of CaV3.3 current relative to WT (black) color coded from green (78%) to red (120%). (c) The number of spikes during positive current injections depends on the magnitude of the current injection (x-axis), but it is only marginally influenced by CaV3.3 density. Black lines represent current density for WT and R/H heterozygous cells, with same color spectrum as in (b), extended to 40% of WT (blue).