Loss of synaptic Zn2+ transporter function increases risk of febrile seizures

Abstract

Febrile seizures (FS) are the most common seizure syndrome and are potentially a prelude to more severe epilepsy. Although zinc (Zn(2+)) metabolism has previously been implicated in FS, whether or not variation in proteins essential for Zn(2+) homeostasis contributes to susceptibility is unknown. Synaptic Zn(2+) is co-released with glutamate and modulates neuronal excitability. SLC30A3 encodes the zinc transporter 3 (ZNT3), which is primarily responsible for moving Zn(2+) into synaptic vesicles. Here we sequenced SLC30A3 and discovered a rare variant (c.892C > T; p.R298C) enriched in FS populations but absent in population-matched controls. Functional analysis revealed a significant loss-of-function of the mutated protein resulting from a trafficking deficit. Furthermore, mice null for ZnT3 were more sensitive than wild-type to hyperthermia-induced seizures that model FS. Together our data suggest that reduced synaptic Zn(2+) increases the risk of FS and more broadly support the idea that impaired synaptic Zn(2+) homeostasis can contribute to neuronal hyperexcitability.

Figure 1. Enrichment of SLC30A3 variant in FS.

(a) Sequence chromatogram showing the c.892C > T SLC30A3 variant enriched in FS patients. (b) Multiple species alignment of ZNT3 protein sequence showing the R298 amino acid is highly conserved (arrow). Rhesus = Rhesus monkey; Prairie = Prairie vole. (c) Schematic showing domain structure of the ZNT3 protein. Light-blue shading indicates domains involved in Zn²⁺ binding. The R298C variant is located in the cytoplasmic domain near the C-terminus. Adapted from.

Figure 2. Pedigrees of probands carrying ZNT3 variant.

All three probands carry the c.892C > T (p.R298C) variant in SLC30A3. The segregation of the variants in their respective pedigrees is shown: *heterozygous c.892C > T (p.R298C) genotype (C/T); wt, homozygous wild-type genotype (C/C).

Figure 3. Functional analysis reveals a loss-of-function due to the ZNT3 (R298C) variant. ai.

Western blot analysis of the three fractions isolated by differential centrifugation including: cell/nuclear (Nucl.), endosomal/mitochondrial (Endo) and synaptic –like microvesicle (SLMV) fractions. Cross-reactivity with synaptophysin (SYP) and vGlut1 antibodies is greatest in the P3 (200,000 g) fraction confirming SLMV enrichment. aii Confocal images of representative PC12 cells transfected with eGFP, ZNT3 (WT)-eGFP and ZNT3 (R298C)-eGFP. Scale bar = 15 μm (b) Median eGFP fluorescence in PC12 cells transfected with ZNT3 (WT) and ZNT3 (R298C) confirming similar ZnT3 translation from both constructs (p = 0.74, n = 4, Student’s paired t-test).(c) Normalized Zn²⁺ concentration in the SLMV fraction of ZNT3 (WT) and ZNT3 (R298C) transfected PC12 cells from four independent experiments. Zn²⁺ was significantly reduced in ZNT3 (R298C) SLMVs (*p = 0.0013, n = 4, Student’s ratio paired t-test) (d) Mean fluorimetry measurements of eGFP in the SLMV fraction of ZNT3 (WT) and ZNT3 (R298C) transfected and in non-transfected PC12 cells. Fluorescence was significantly reduced in ZNT3 (R298C) relative to ZNT3 (WT) and was indistinguishable from auto-fluorescence in non-tranfected cells (*p = 0.0001, n = 3 for all groups, One way ANOVA).

Figure 4. ZnT3 null mice have heightened sensitivity to heat-induced clonic-tonic seizures.

Kaplan-Meier curves showing time to first tonic-clonic seizure during exposure to heat for wild-type and ZnT3 knock-out mice homozygous are significantly different (p = 0.01, wild-type, n = 8; ZnT3, n = 11; Mantel-Cox test).