Functional consequences of calmodulin variants identified among schizophrenia patients and controls

Figures

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  Fig. 1. CALM1-3 protein variants in cardiac and SCZ patients are enriched in the calmodulin C-lobe. Three genes, CALM1-3, produce identical calmodulin proteins, represented by the three rows. Numbering follows the immature 149 amino acid sequence, although the initial methionine is cleaved off during translation. Each circle represents an independent case of a calmodulin variant carrier. Circles below the sequences represent patients reported in the ICalmR, and circles above represent patients identified in the present study. Colors of the circles reflect carriers with cardiac arrhythmia (yellow), SCZ (red), or control carriers (teal), with the associated amino acid position and substitution indicated. Carriers from the ICalmR with primary neurological features are indicated with a black circle. The C-lobe of calmodulin, harboring the majority of cardiac and all SCZ variants, is framed with a dashed square. Amino acids that coordinate calcium are highlighted in dark gray. ICalmR: International Calmodulinopathy Registry. LQTS: Long QT syndrome. SCZ: schizophrenia.

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  Fig. 2. High levels of CALM1-3 mRNA in the brain. A, B mRNA levels of CALM1-3, CLDN4 (negative control), UBC (positive control), CACNA1C (encodes CaV1.2), and ten genes for which rare genetic variants are associated with increased SCZ risk [40]. A Transcript levels extracted from the genotype-tissue expression (GTEx) project. B Single-cell spatial transcriptomics data from human hippocampal fetuses at gestational weeks 16-27 [43] (see also Figs. S2–4). C In situ hybridization with RNAscope probes (red signal) targeting the indicated genes of a healthy human donor. Arrows indicate examples of positively stained neuronal cell bodies, and arrowheads indicate examples of stained neuronal processes. The images are representative of three individual stains of two donors (see also Fig. S5). Scale bars are 2 mm and 100 µm for insets. CaV1.2: Voltage-gated calcium channel isoform 1.2. ExN(DG): Dentate gyrus excitatory neurons. ExN(non-DG): Non-dentate gyrus excitatory neurons. InN(CGE): Caudal ganglion eminence-derived inhibitory neurons. InN(MGE): Medial ganglion eminence-derived inhibitory neurons. OPC: oligodendrocyte progenitor cells. SCZ: schizophrenia. TP10K: Transcripts per 10,000. TPM:…

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  Fig. 3. Calmodulin SCZ variants significantly affect calcium binding. A Visualization of calmodulin amino acid positions affected in LQTS patients, SCZ patients, and in control individuals on the three-dimensional structure of calmodulin (gray) in apo-state (PDB: 1DMO) or with calcium ions (black) bound (PDB: 1CLL). B Calcium affinities of the indicated N-lobe calmodulin variants. C Calcium affinities of the indicated C-lobe calmodulin variants. Data in B and C are fitted from binding curves in Fig. S6 and detailed in Table S3-4, n = 3-11 individual experiments. *P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 with one-way ANOVA and Dunnett’s post hoc test. D Summarized effect on calcium binding in SCHEMA carriers of calmodulin variants. Statistics: Fisher’s exact test. Yellow, LQTS; red, SCZ; teal, controls. Ctrl, control; LQTS, long QT syndrome; KD, dissociation constant; SCZ, schizophrenia.

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  Fig. 4. Calmodulin variants in SCHEMA primarily affect the apo structure. Circular dichroism was used to determine structural effects of the indicated variants on the α-helices of apo-calmodulin (A) or calcium-bound calmodulin (B) as well as the calcium-induced difference (C) between the two states (n = 4-16 individual experiments). Data for D130G and Q136P is reproduced from a previous study [63]. *P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 with one-way ANOVA with Dunnett’s post hoc test. Raw spectra are shown in Fig. S7-8, and summarizing statistics are in Table S5. Yellow, LQTS; red, SCZ; teal, controls. LQTS: long QT syndrome. SCZ: schizophrenia.

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  Fig. 5. Calmodulin SCZ and control variants have different effects on calcium-dependent CaV1.2-IQ binding. A Schematic of calcium-dependent calmodulin (CaM) binding to the IQ-domain of CaV1.2 (CaV1.2-IQ). B Binding affinity, represented by the dissociation constant, KD, of calmodulin binding to CaV1.2-IQ as a function of free calcium concentration. Vertical gray shades indicate data presented in detail in C-E. C-E Calmodulin:CaV1.2-IQ affinity at 50 nM, 200 nM, and 800 nM free calcium, representing three different physiological conditions. F EC50 values reflecting the calcium sensitivity of calmodulin when bound to the CaV1.2-IQ domain. Data for D130G, Q136P, and F142L is reproduced from previous studies [62, 63]. n = 3-10 individual experiments for B-F, *P < 0.05, *** P < 0.001, **** P < 0.0001 with one-way ANOVA followed by Dunnett’s post hoc test. Statistical calculations for B-F are detailed in Table S6-7, and raw data is shown in Fig S9-11. G Schematic summary of calmodulin variant effects on calcium binding and calcium-dependent CaV1.2-IQ binding. Yellow, LQTS; red, SCZ; teal, controls. EC50, half maximal effective concentration. CaV1.2: voltage-gated calcium channel 1.2. GoF:…

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  Fig. 6. Small effects of calmodulin variants from SCZ patients on CDI of the CaV1.2 channel. A Calmodulin binds the C-terminal tail of CaV1.2 and regulates channel closure in a process termed calcium dependent inactivation (CDI). B-I Top panels show exemplar currents evoked by 30-mV voltage step in cells co-transfected with CaV1.2 and the indicated calmodulin variants. CDI manifests as the rapid decay in calcium current (colored trace) as compared to barium (black trace). For comparison, the trace for WT calmodulin is reproduced in gray behind the exemplar traces for the indicated variants. Bottom panels show population data for CDI across voltages. r300 measures the current remaining after 300 ms, after normalization to peak current. Data in H and I is reproduced from a previous study along with the corresponding WT in gray [18]. J Quantification of f300, the difference between calcium and barium at 30 mV, after normalization by the barium r300 value. n = 6-16 individual experiments, ** P < 0.01 with one-way ANOVA followed by Dunnett’s post hoc test. Statistical calculations are detailed in Table S8. CaV1.2: voltage-gated calcium channel 1.2. SCZ: schizophrenia.