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

CACNA1I is a candidate schizophrenia risk gene. It encodes the pore-forming human CaV3.3 α1 subunit, a subtype of voltage-gated calcium channel that contributes to T-type currents. Recently, two de novo missense variations, T797M and R1346H, of hCaV3.3 were identified in individuals with schizophrenia. Here we show that R1346H, but not T797M, is associated with lower hCaV3.3 protein levels, reduced glycosylation, and lower membrane surface levels of hCaV3.3 when expressed in human cell lines compared to wild-type. Consistent with our biochemical analyses, whole-cell hCaV3.3 currents in cells expressing the R1346H variant were ~50% of those in cells expressing WT hCaV3.3, and neither R1346H nor T797M altered channel biophysical properties. Employing the NEURON simulation environment, we found that reducing hCaV3.3 current densities by 22% or more eliminates rebound bursting in model thalamic reticular nucleus (TRN) neurons. Our analyses suggest that a single copy of Chr22: 39665939G > A CACNA1I has the capacity to disrupt CaV3.3 channel-dependent functions, including rebound bursting in TRN neurons, with potential implications for schizophrenia pathophysiology.


Reverse transcription and quantitative PCR.
Total RNA from each cell line was harvested using RNAeasy Plus (Qiagen). 10 μ g of total RNA was reverse-transcribed and cDNA synthesized using random hexamer priming (Transcriptor cDNA synthesis, Roche). FastStart Universal SYBR Green 2X Master Mix (Roche) was used to perform quantitative PCR for hCa V 3.3 (CACNA1I: 5′ CAATGGACTGGATGCTGTTG/5′ ATCCAGGGGTTGT GGTTG) and β -actin (ACTB: 5′ CCAACCGCGAGAAGATGA/5′ CCAGAGGCGTACAGGGATAG). CACNA1I mRNA in each cell line was analyzed by the relative quantitation of gene expression method and using ACTB that encodes β -actin as the reference control gene (Δ Δ Ct method) 33 . Threshold amplification cycle (CT) values were obtained for target (CACNA1I) and internal control (ACTB) to calculate ∆ CT (CT target-CT reference), and Δ Δ Ct calculated before and after induction of CACNA1I by doxycycline treatment. We carried out 3-4 technical replicates, from three independent cell culture and dox-induction step (biological triplicates). The biological variability in our RT-qPCR experiments stems primarily from different overall levels of mRNA induction across biological replicates.
The protein measurements for T797M and R1346H channels have similar dispersion, and scales proportionally to the absolute values. The coefficient of variation (CV) for whole cell protein level for R1346H is 40% compared to 31% for T797M. Using 1000 bootstrapping samples we estimated that the 95% confidence interval for T797M CV is (0.21,0.42), while the 95% CI for R1346H CV is (0.26, 0.54). The 95% confidence interval for the difference in CV between T797M and R1346H is (− 0.26, 0.09)-not different from 0-indicating that the data dispersion between T797M and R1346H is statistically equivalent.
Scientific RepoRts | 6:34233 | DOI: 10.1038/srep34233 Deglycosylation. Deglycosylation was performed on input or eluate samples with Protein Deglycosylation Mix (New England Biolabs). Briefly, 60 μ g of total protein (input) or biotin labeled membrane protein (eluate) was denatured in Glycoprotein Denaturing Buffer for 20 minutes at RT. The denatured protein was then treated with either buffer alone (control) or with the Deglycosylation Enzyme Cocktail (PNGase F, O-Glycosidase, Neuraminidase, Galactosidase, and β -N-acetylglucosaminidase) for 1 hour at 37 °C in a solution of 50 mM sodium phosphate and 1% NP-40. Samples were analyzed by immunoblotting.
Conventional electrophysiology. Voltage-gated calcium currents (Ca V ) were only resolved in Flp-In T-REx HEK293 cells after, and not before, doxycycline induction of wild-type or mutant hCa V 3.3 cDNA. Whole-cell patch clamp recordings were used to compare hCa V 3.3 channel currents in doxycycline treated Flp-In T-REx HEK293 cells carrying a single copy of exogenous wild-type or mutant hCa V 3.3 cDNA. Recordings were performed as previously reported 35,36 . The macroscopic Ca V 3.3 currents recorded in this expression system originate from the gating of Ca V 3.3 channels: we have never recorded Ca V currents in untransfected tsA201, HEK293 or Flp-In T-REx HEK293 cells under the recording conditions used in this study (see also refs 35,37-39). Wild-type macroscopic Ca V 3.3 currents shown here have all the properties of Ca V 3.3 currents and the peak current is about 60 pA/pF, equivalent to ~ 700-750 pA, with only 2 mM calcium as the charge carrier. The properties of the macroscopic as well as single Ca V 3.3 channel currents are completely consistent with previously published data e.g. 2,40 .
Whole-cell external recording solution contained: 2 mM CaCl 2 , 10 mM HEPES, 140 mM NaCl, pH adjusted to 7.2 with NaOH and the intracellular pipette solution contained: 126 mM CsCl, 10 mM EGTA, 1 mM EDTA, 10 mM HEPES, 4 mM MgATP, pH 7.2 with CsOH. Whole-cell hCa V 3.3 currents were evoked by square step depolarizations from a holding potential of − 100 mV. Currents were leak subtracted online using a P/− 4 protocol. Currents recorded with Axopatch 200B amplifier (Molecular Devices, LLC) were sampled at 20 kHz and filtered at 2 kHz. For single-channel recordings, we used the HEK293-derived cell line tsA201 transiently expressing wild-type or mutant hCa V 3.3 cDNA for 48 hrs. We used a pipette solution for single channel recording which is optimized to isolate Ca V currents: 110 mM BaCl 2 , 2 mM CsCl, 10 mM HEPES, pH adjusted to 7.2 with Ba(OH) 2 and the extracellular bath solution contained: 135 mM potassium aspartate, 10 mM EGTA, 5 mM HEPES, 5 mM KCl, pH adjusted to 7.2 with KOH (see refs 35,37-39). The high potassium extracellular solution effectively clamps the membrane potential to 0 mV eliminating contributions of the membrane potential to the transmembrane patch voltage. To reduce electrode capacitance in single channel recording, pipettes were coated with Sylgard 184 (Dow Corning, Co) and polished to resistances of 5-8 MΩ. Single channel currents recorded using Axopatch 200B amplifier (Molecular Devices, LLC) were sampled at 10-20 kHz and filtered at 1 kHz. Clampfit10 software (Molecular Devices, LLC) was used for single channel analyses. Leak subtraction was performed offline using a noiseless stimulated null trace. All recordings were obtained at room temperature.
Properties of single Ca V 3.3 channel currents. The estimated reversal potential for current flow through single Ca V 3.3 channels was positive to 0 mV, the single channel conductance estimated from the single channel current voltage relationship was consistent with single channel conductance measurements for Ca V 3, and Ca V 3.3 specifically e.g 2,40 ; single Ca V channel currents exhibited classic slow closing kinetics during the repolarization step 2 ; and we have never resolved single Ca V channel currents in tsA201 cells in un-transfected cells 35,37 . Experimental Design. All data were acquired without knowledge of clone identity, all analyses were done before revealing clone identity, and the experimenter interleaved recordings from cells expressing different clones on each experimental day. To compare current amplitudes across cells, we converted to current density (pA/pF) to normalize for cell size. In high throughput electrophysiology, cell capacitance is not measured but the much larger sample sizes (> 100) add statistical power and compensate for the relatively small variation in size among HEK293 cells. Peak currents evoked by depolarizing pulses were used to generate current voltage relationships. These were fit with the sum of a Goldmann-Hodgkin-Katz flux equation (φ ) and an exponential function to model the shape of inward and outward unitary conductance, multiplied by a single Boltzmann function to summarize gating.
High throughput electrophysiology. Recordings from doxycycline-induced Flp-In T-REx HEK293 cells expressing hCav3.3 channels were performed using Ion-works Barracuda by ChanTest, Charles River 41 . Briefly, cells were harvested, washed and re-suspended in HEPES-buffered physiological saline before adding to the single-hole 384-well patch plate. External recording solution contained: 137 mM NaCl, 4 mM KCl, 7 mM CaCl 2 , 1 mM MgCl 2 , 10 mM HEPES, and 10 mM glucose, pH adjusted to 7.4 with NaOH. The internal solution contained 90 mM CsF, 50 mM CsCl, 2 mM MgCl 2 , 0.5 mM EGTA, and 10 mM HEPES, pH 7.2 adjusted with CsOH. Membrane currents were recorded with on-board patch clamp amplifiers, after establishing whole-cell configuration using amphotericin B (100 μ g/ml). Peak current amplitudes measured from cells expressing hCa V 3.3 WT, T797M and R1346H were fit to a bimodal distribution comprising two log-normal functions (sub-distributions) of different scale and shape factors. Descriptors of interest, the Bernoulli parameter and the median of the larger sub-distribution were determined by finding the parameters that maximize the likelihood of the data (maximum-likelihood estimates). To determine confidence intervals, a bootstrap procedure was used: the fitting algorithm was repeated for each resampling of the data.

hCa V 3.3 expression is disrupted by R1346H.
We set out to test if two rare schizophrenia risk variations, identified in CACNA1I from exome sequencing of trio samples 19 , disrupt Ca V 3.3 channel function as assessed in HEK cell line expression systems. Figure 1a,b illustrate the approximate location of amino acids T797 and R1346 in putative extracellular loops that link transmembrane helices 5 and 6 in domains II and III, respectively. We used Flp-In T-REx HEK293 cells that harbor inducible, single-copy stable integration of cDNAs encoding C-terminus FLAG-tagged WT, T797M, or R1346H, to investigate hCa V 3.3 function. These cells do not express endogenous Ca V 3.3 channels. The anti-FLAG Ca V 3.3 signal was only induced following exposure to doxycycline ( WT hCa V 3.3-FLAG protein isolated from whole cell lysates migrates as a doublet at ~250 kDa and > 250 kDa (Fig. 2a). The > 250 kDa band is glycosylated based on the shift to ~250 kDa after exposing cell lysates to glycosidases (Fig. 2b). We compared total hCa V 3.3-FLAG protein levels in lysates from cells expressing WT, T797M and R1346H and found that the hCa V 3.3-FLAG signal in cells expressing R1346H was reduced significantly compared to WT (Fig. 2c, compare lanes 1 and 3; Fig. 2d Whole cell, F(2,53) = 89.43, p < 0.0001, one-way ANOVA followed with Dunnett's test, p WT,R1346H < 0.0001). The relative level of the > 250 kDa glycosylated hCa V 3.3 signal, as a fraction of the total hCa V 3.3 signal, was significantly reduced in cells expressing R1346H compared to WT controls ( Fig. 2f, F (2,23) = 10.04, p = 0.014, one-way ANOVA with Dunnett's post test, p WT, R1346H = 0.0439). By contrast, the total hCa V 3.3 signal, and the relative abundance of the glycosylated > 250 KDa signal from cells expressing T797M were not significantly different from WT (Fig. 2c, lanes 1 and 2; Fig. 2d, whole cell, one-way ANOVA followed with Dunnett's test, p WT,T797M = 0.2491; Fig. 2f, one-way ANOVA and Dunnett's test, p WT, T797M = 0.8318).
Our data demonstrated that hCa V 3.3 protein levels are substantially lower in cells expressing R1346H compared to WT and T797M. The glycosylated R1346H hCa V 3.3 protein fraction (> 250 kDa) is preferentially reduced relative to WT (Fig. 2f), suggesting that R1346H might interfere with glycosylation and surface trafficking of hCa V 3.3.
To compare rates of hCa V 3.3 protein decay in cells expressing WT, T797M and R1346H, we treated cells with 0.8 μ g/mL puromycin to inhibit protein translation and measured levels of hCa V 3.3 protein at different time points. At this concentration and duration of exposure, puromycin had no obvious cellular toxicity 43,44 . The 250 kDa lower molecular weight hCa V 3.3 signal decayed relatively rapidly after protein translation was inhibited and was not detectable after 6 hours of puromycin exposure (Fig. 3a). By contrast, the glycosylation-associated > 250 kDa hCa V 3.3 signal decayed more slowly and incompletely (Fig. 3a,b). After 48 hrs of exposure to puromycin, the > 250 kDa hCa V 3.3 signal was reduced ~50% of control levels similarly among three genotypes (Fig. 3b, 48 hr data point, F (2,20) = 2.675, p = 0.0935, one-way ANOVA with Dunnett's post test, p WT, R1346H = 0.259, p WT, T797M = 0.540). In the first 2 hrs of puromycin treatment, the > 250 kDa hCa V 3.3 signal increased ~40% and ~30% in cells expressing WT and T797M respectively and the levels were not different between the two conditions ( Fig. 3b, 2 hr data point, F (2,20) = 17.6, p < 0.001, one-way ANOVA with Dunnett's post test, p WT, T797M = 0.859). We observed ~15% reduction in the > 250 kDa band in cells expressing R1346H following exposure to puromycin, and the overall protein levels were lower compared to WT (Fig. 3b, 2 hr data point, F (2,20) = 17.6, p < 0.001, one-way ANOVA with Dunnett's post test, p WT, R1346H = 0.0154). We also showed that GAPDH levels did not decrease following puromycin treatment in cells expressing different Ca V 3.3 clones over the time course of the experiment (Fig. 3c). Our data suggest that R1346H reduces the net accumulation of glycosylated hCa V 3.3 compared to wild-type and does not impact the rates of channel protein decay.
Collectively, our data are consistent with the hypothesis that R1346H interferes with surface trafficking of hCa V 3.3 by a mechanism that may involve glycosylation. Two putative N-linked glycosylation sites are within 1 and 4 amino acids of R1346 (N1345 and N1342). Mutations of putative glycosylation sites, N192Q and N271Q in S3-S4 and S5-S6 linkers of domain I of Ca V 3.2 protein are associated with reduced levels of biotinylated, functional channels on the cell membrane surface 45 . The glycosylation states of auxiliary subunits of Ca V channels have also been reported to influence protein function [46][47][48] . T797M and R1346H affect the functional properties of hCa V 3.3 channels using whole-cell recording from the same Flp-In T-REx HEK293 cells expressing FLAG-tagged hCa V 3.3 as described above. We used 2 mM Ca 2+ as the charge carrier for conventional whole-cell recording and resolved large Ca V 3.3 currents that peaked at ~60 pA/pF in cells expressing WT channels. Consistent with our biochemical analyses, hCa V 3.3 channel current densities in doxycycline-induced HEK293 cells expressing R1346H, but not T797M, were smaller relative to those in cells expressing WT hCa V 3.3, independent of membrane voltage (Fig. 4a). To compare hCa V 3.3 currents across a range of voltages, we estimated permeability rates from Boltzmann-GHK fits of individual current voltage data sets from cells expressing WT, R1346H and T797M hCa V 3.3 ( Fig. 4b; left). Permeability rate was used as a measure of the overall current flow in a cell for a range of voltages (see methods). Permeability rates were ~43.6% lower for hCa V 3.3 currents in cells expressing R1346H compared to WT ( Fig. 4b; left, p WT,R1346H = 0.00054, Kolmogorov-Smirnov test followed by Bonferroni correction), whereas permeability rates calculated from cells expressing T797M were similar to WT ( Fig. 4b; left, p WT,T797M = 0.923, Kolmogorov-Smirnov test followed by Bonferroni correction). Estimates of the membrane potential at which Ca V 3.3 currents reverse direction were not different in cells expressing WT, R1346H and T797M ( Fig. 4b; right, p WT,R1346H = 0.36, p WT,T797M = 0.74, Kolmogorov-Smirnov test followed by Bonferroni correction) suggesting that ion selectivity in hCa V 3.3 channels is unchanged by R1346H and T797M.
We next contracted ChanTest to perform unbiased, high throughput electrophysiology using the Flp-In T-REx HEK293 cells expressing hCa V 3.3 channels, and data are shown in Fig. 4c (IonWorks Barracuda; Molecular Devices). The large sample sizes, possible from high throughput automated whole-cell analyses, allowed for population analyses of hCav3.3 current densities in cells expressing WT, R1346H or T797M (Fig. 4a,b). Population data, displayed in bee swarm and cumulative frequency plots, illustrate that hCa V 3.3 currents in cells expressing R1346H hCa V 3.3 are on average 2-fold smaller compared to WT (Fig. 4c). The cumulative frequency relationships for each condition were bimodal, consistent with two populations of Flp-In T-REx HEK293 cells: one expressing and a smaller fraction not expressing hCa V 3.3 currents (Fig. 4c). Parameterization of each distribution, allowed by the larger sample size, showed that the percentage of cells not expressing hCa V 3.3 current was similar among all variants (WT: 16%, T797M: 16%, R1346H: 20%) and that the median hCa V 3.3 current of cells expressing R1346H was ~2-fold relative to WT (Fig. 4c, p WT,R1346H < 0.0001, Kolmogorov-Smirnov test followed by Bonferroni correction). These data are consistent with our findings from conventional whole-cell recording.
Lower hCa V 3.3 current densities in cells expressing R1346H could originate from fewer hCa V 3.3 channels on the cell surface, from reduced current flow through individual hCa V 3.3 channels, when open, or a combination of both. We used high-resolution, low-noise cell-attached patches to measure the rate of ion flow through single hCa V 3.3 channels directly (Fig. 5a-c). The amplitude of single hCa V 3.3 channel currents was consistent with that reported previously for single Ca V 3.3 currents 2 and indistinguishable among WT, R1346H, and T797M hCa V 3.3 recordings over a range of test potentials (~13 pS, 110 mM barium as charge carrier; Fig. 5c, p WT,R1346H = 0.85, and p WT,T797M = 0.53, Kolmogorov-Smirnov test followed by Bonferroni correction). We measured single channel current amplitudes from tail currents to generate the single channel I-V relationship because of the larger current amplitudes (greater driving force) at negative voltages. The slow gating kinetics typical of Ca V 3.3 currents is illustrated in Fig. 5 from individual traces as well as captured in the ensemble averages. We conclude that the smaller Ca V 3.3 current densities in cells expressing R1346H reflect reduced numbers of Ca V 3.3 channels on the cell surface relative to WT, but the amount of current that flows through individual hCa V 3.3 channels is unaffected by R1346H.
We completed our assessment of R1346H and T797M hCa V 3.3 channel properties not captured by analyses of peak current-voltage relationships, using a series of voltage protocols to evaluate whole-cell currents. We assessed: voltage-dependence from tail current analyses (Fig. 6a- Kolmogorov-Smirnov test followed by Bonferroni correction), kinetics of channel activation from − 50 to 20 mV voltages (Fig. 6d,e), rate of channel closing as derived from the time constant of the tail current decay (Fig. 6f. τ closing at-60 mV, p WT,R1346H = 0.43, p WT,T797M = 0.97, Kolmogorov-Smirnov test followed by Bonferroni correction), voltage-dependence of channel inactivation (Fig. 7a,b. V 1/2 , p WT,R1346H = 0.12, p WT,T797M = 0.76. Slope (k), p WT,R1346H = 0.52, p WT,T797M = 0.19, Kolmogorov-Smirnov test followed by Bonferroni correction), and time course of channel inactivation from − 50 to 0 mV (Fig. 7c,d). Tail current kinetics provide a measure of the overall rate of channel closing, because the tail potential is below the threshold for channel opening and the slow closing kinetics is a hallmark feature of Ca V 3 channels, compared to other Ca V channels. We conclude from these extensive analyses, that R1346H and T797M do not affect the biophysical properties of hCa V 3.3 channels as assessed in human cell lines, including the time course of recovery from inactivation (T797M; data not shown). It is important to note that our analyses, which find T797M has no measurable effect on the basic features of hCa V 3.3 channels including expression levels and biophysical properties, do not rule out a potential effect of T797M that depends on the presence of cofactors in the native environment.   (Fig. 8a). This phenomenon is mediated by activation of dendritic Ca V 3.3 channels that are recruited from a previously inactivated state when the membrane is hyperpolarized. Ca V 3.3 underlies 90% of the low threshold, voltage-gated calcium channel expressed in TRN 9 and rebound bursting is absent in TRN neurons of mice lacking Ca V 3.3 6,9 . By simulating TRN neuron excitability in the NEURON environment 7 we show that: (i) rebound bursting is highly sensitive to Ca V 3.3 channel density; (ii) rebound bursting is eliminated when Ca V 3.3 channel density is reduced to 78% or less of initial WT values ( Fig. 8b; black line shows WT relationship); and (iii) firing of TRN neurons evoked by depolarizing current injections is insensitive to changes in dendritic Ca V 3.3 current densities to 40% of initial WT values (Fig. 8a,c).
To simulate heterozygosity-equal contribution of WT and R1346H alleles-we reduced Ca V 3.3 current density to 72.5% of WT levels and showed that this reduction in Ca V 3.3 current density fails to support rebound bursting regardless of hyperpolarization magnitude (Fig. 8a, top three rows). In contrast, firing of TRN neurons, evoked by depolarizing current injections is unaffected by R1346H (Fig. 8a, bottom two rows). The results from NEURON simulation are consistent with the notion that Depolarization-induced firing is primarily mediated by activation of voltage-gated ion channels other than Ca V 3.3 in TRN 8,9 . Discussion CACNA1I has been identified as a candidate schizophrenia risk gene based on genome wide association studies, and on the identification of de novo, rare missense variations in CACNA1I from exome sequencing of schizophrenia proband trios 19,31 . Our study is the first to assess the functional impact of two missense CACNA1I variants found in schizophrenia patients, but not in unaffected family members 31 . Based on our analyses, we find that the de novo coding variant Chr22: 39665939G > A of CACNA1I 31 , is sufficiently disruptive to Ca V 3.3 in the heterozygous condition to impact rebound bursting in a TRN model neuron. Our data lend support to the proposal that Ca V 3.3 R1346H, which is proposed as one of several de novo risk variations that contribute to schizophrenia pathophysiology 31 , is damaging. We did not find any evidence that the de novo coding variation Chr22: 39659492C > T of CACNA1I (T797M Ca V 3.3) is disruptive in biochemical and electrophysiological HEK cell assays. However, we cannot rule out the possibility that T797M impacts Ca V 3.3 channel function by mechanisms that are not reconstituted in HEK cells.
Our findings are interesting in light of documented functional associations between reduced Ca V 3.3 expression in TRN neurons, rebound bursting of TRN neurons, sleep spindle oscillations, and sleep spindle coherence across cortex in schizophrenia 6,10,14,[49][50][51] . Moreover, reduced spindle activity is a heritable component of the sleep electroencephalogram patterns detected in the 1 st degree relatives of people with schizophrenia 52 .
Our data are consistent with a mechanism by which R1346H interferes with Ca V 3.3 glycosylation and plasma membrane trafficking leading to reduced Ca V 3.3 current density. An extracellular N-linked glycosylation motif N1345 (N-x-S/T) encompasses R1346 residue and glycosylation of proteins in the endoplasmic reticulum (ER) is known to influence the rate of protein transport from the ER through the Golgi apparatus to the plasma membrane 53 . Our studies add to reports that glycosylation of many ion channel proteins, including Ca V 3.2, regulates their levels of surface expression 45,[54][55][56][57][58] . Several human diseases are known to arise from defects in glycosylation. For example, in cystic fibrosis causal mutations in human CFTR lead to altered glycosylation patterns of CFTR channel protein and channel trafficking defects in lung epithelial cells, and in long QT syndrome, coding mutations in human KCNE and KCNQ lead to reduced potassium ion channel glycosylation and reduced expression in the heart 59-62 .
Ca V 3.3 is essential for rebound bursting in TRN neurons, and we show that reduced Ca V 3.3 current density in model TRN neurons expressing R1346H is sufficient to disrupt this phenomenon. Ca V 3.3 is expressed in other brain regions including the cortex and in mitral cell dendrites of the olfactory bulb 63 . In mitral cells, Ca V 3.3 contributes to modulation of evoked and asynchronous release, and it mediates rebound bursting 63 . Deficits in olfaction have, for several years, been described in people with schizophrenia 64 .
It is widely accepted that schizophrenia disease risk depends on the accumulated effect of multiple or many common risk loci 19 , but the relative contribution of each individual rare variant to the disease risk is not known 17 . Rare, coding variations have the greatest potential to disrupt protein function and are likely to make greater contribution to complex common disease risk including schizophrenia 17,19,25,30,65 . Our experiments were designed to assess the functional consequences of T797M and R1346H on Ca V 3.3 channel activity in a robust expression system, and they demonstrate that R1346H disrupts Ca V 3.3 channel trafficking to the plasma membrane. We did 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 not identify a phenotype associated with T797M in our assays of Ca V 3.3 function and, as discussed above, we cannot rule out the possibility that T797M will be disruptive to Ca V 3.3 signaling in the native environment, but our analyses should help guide future studies designed to assess the potential contribution of R1346H-Ca V 3.3 to schizophrenia risk .