Ca2+ sensor proteins in spontaneous release and synaptic plasticity: Limited contribution of Doc2c, rabphilin-3a and synaptotagmin 7 in hippocampal glutamatergic neurons.

Presynaptic neurotransmitter release is strictly regulated by SNARE proteins, Ca2+ and a number of Ca2+ sensors including synaptotagmins (Syts) and Double C2 domain proteins (Doc2s). More than seventy years after the original description of spontaneous release, the mechanism that regulates this process is still poorly understood. Syt-1, Syt7 and Doc2 proteins contribute predominantly, but not exclusively, to synchronous, asynchronous and spontaneous phases of release. The proteins share a conserved tandem C2 domain architecture, but are functionally diverse in their subcellular location, Ca2+-binding properties and protein interactions. In absence of Syt-1, Doc2a and -b, neurons still exhibit spontaneous vesicle fusion which remains Ca2+-sensitive, suggesting the existence of additional sensors. Here, we selected Doc2c, rabphilin-3a and Syt-7 as three potential Ca2+ sensors for their sequence homology with Syt-1 and Doc2b. We genetically ablated each candidate gene in absence of Doc2a and -b and investigated spontaneous and evoked release in glutamatergic hippocampal neurons, cultured either in networks or on microglial islands (autapses). The removal of Doc2c had no effect on spontaneous or evoked release. Syt-7 removal also did not affect spontaneous release, although it altered short-term plasticity by accentuating short-term depression. The removal of rabphilin caused an increased spontaneous release frequency in network cultures, an effect that was not observed in autapses. Taken together, we conclude that Doc2c and Syt-7 do not affect spontaneous release of glutamate in hippocampal neurons, while our results suggest a possible regulatory role of rabphilin-3a in neuronal networks. These findings importantly narrow down the repertoire of synaptic Ca2+ sensors that may be implicated in the spontaneous release of glutamate.

Doc2 Proteins Are Not Required for the Increased Spontaneous Release Rate in Synaptotagmin-1-Deficient Neurons.

Regulated secretion is controlled by Ca2+ sensors with different affinities and subcellular distributions. Inactivation of Syt1 (synaptotagmin-1), the main Ca2+ sensor for synchronous neurotransmission in many neurons, enhances asynchronous and spontaneous release rates, suggesting that Syt1 inhibits other sensors with higher Ca2+ affinities and/or lower cooperativities. Such sensors could include Doc2a and Doc2b, which have been implicated in spontaneous and asynchronous neurotransmitter release and compete with Syt1 for binding SNARE complexes. Here, we tested this hypothesis using triple-knock-out mice. Inactivation of Doc2a and Doc2b in Syt1-deficient neurons did not reduce the high spontaneous release rate. Overexpression of Doc2b variants in triple-knock-out neurons reduced spontaneous release but did not rescue synchronous release. A chimeric construct in which the C2AB domain of Syt1 was substituted by that of Doc2b did not support synchronous release either. Conversely, the soluble C2AB domain of Syt1 did not affect spontaneous release. We conclude that the high spontaneous release rate in synaptotagmin-deficient neurons does not involve the binding of Doc2 proteins to Syt1 binding sites in the SNARE complex. Instead, our results suggest that the C2AB domains of Syt1 and Doc2b specifically support synchronous and spontaneous release by separate mechanisms. (Both male and female neurons were studied without sex determination.)SIGNIFICANCE STATEMENT Neurotransmission in the brain is regulated by presynaptic Ca2+ concentrations. Multiple Ca2+ sensor proteins contribute to synchronous (Syt1, Syt2), asynchronous (Syt7), and spontaneous (Doc2a/Doc2b) phases of neurotransmitter release. Genetic ablation of synchronous release was previously shown to affect other release phases, suggesting that multiple sensors may compete for similar release sites, together encoding stimulus-secretion coupling over a large range of synaptic Ca2+ concentrations. Here, we investigated the extent of functional overlap between Syt1, Doc2a, and Doc2b by reintroducing wild-type and mutant proteins in triple-knock-out neurons, and conclude that the sensors are highly specialized for different phases of release.

Synaptotagmin-1 and Doc2b Exhibit Distinct Membrane-Remodeling Mechanisms.

Synaptotagmin-1 (Syt1) is a calcium sensor protein that is critical for neurotransmission and is therefore extensively studied. Here, we use pairs of optically trapped beads coated with SNARE-free synthetic membranes to investigate Syt1-induced membrane remodeling. This activity is compared with that of Doc2b, which contains a conserved C2AB domain and induces membrane tethering and hemifusion in this cell-free model. We find that the soluble C2AB domain of Syt1 strongly affects the probability and strength of membrane-membrane interactions in a strictly Ca2+- and protein-dependent manner. Single-membrane loading of Syt1 yielded the highest probability and force of membrane interactions, whereas in contrast, Doc2b was more effective after loading both membranes. A lipid-mixing assay with confocal imaging reveals that both Syt1 and Doc2b are able to induce hemifusion; however, significantly higher Syt1 concentrations are required. Consistently, both C2AB fragments cause a reduction in the membrane-bending modulus, as measured by a method based on atomic force microscopy. This lowering of the energy required for membrane deformation may contribute to Ca2+-induced fusion.

Doc2b synchronizes secretion from chromaffin cells by stimulating fast and inhibiting sustained release.

Synaptotagmin-1 and -7 constitute the main calcium sensors mediating SNARE-dependent exocytosis in mouse chromaffin cells, but the role of a closely related calcium-binding protein, Doc2b, remains enigmatic. We investigated its role in chromaffin cells using Doc2b knock-out mice and high temporal resolution measurements of exocytosis. We found that the calcium dependence of vesicle priming and release triggering remained unchanged, ruling out an obligatory role for Doc2b in those processes. However, in the absence of Doc2b, release was shifted from the readily releasable pool to the subsequent sustained component. Conversely, upon overexpression of Doc2b, the sustained component was largely inhibited whereas the readily releasable pool was augmented. Electron microscopy revealed an increase in the total number of vesicles upon Doc2b overexpression, ruling out vesicle depletion as the cause for the reduced sustained component. Further experiments showed that, in the absence of Doc2b, the refilling of the readily releasable vesicle pools is faster, but incomplete. Faster refilling leads to an increase in the sustained component as newly primed vesicles fuse while the [Ca(2+)]i following stimulation is still high. We conclude that Doc2b acts to inhibit vesicle priming during prolonged calcium elevations, thus protecting unprimed vesicles from fusing prematurely, and redirecting them to refill the readily releasable pool after relaxation of the calcium signal. In sum, Doc2b favors fast, synchronized release, and limits out-of-phase secretion.

DOC2 isoforms play dual roles in insulin secretion and insulin-stimulated glucose uptake.

AIMS/HYPOTHESIS: Glucose-stimulated insulin secretion (GSIS) and insulin-stimulated glucose uptake are processes that rely on regulated intracellular vesicle transport and vesicle fusion with the plasma membrane. DOC2A and DOC2B are calcium-sensitive proteins that were identified as key components of vesicle exocytosis in neurons. Our aim was to investigate the role of DOC2 isoforms in glucose homeostasis, insulin secretion and insulin action. METHODS: DOC2 expression was measured by RT-PCR and western blotting. Body weight, glucose tolerance, insulin action and GSIS were assessed in wild-type (WT), Doc2a (-/-) (Doc2aKO), Doc2b (-/-) (Doc2bKO) and Doc2a (-/-)/Doc2b (-/-) (Doc2a/Doc2bKO) mice in vivo. In vitro GSIS and glucose uptake were assessed in isolated tissues, and exocytotic proteins measured by western blotting. GLUT4 translocation was assessed by epifluorescence microscopy. RESULTS: Doc2b mRNA was detected in all tissues tested, whereas Doc2a was only detected in islets and the brain. Doc2aKO and Doc2bKO mice had minor glucose intolerance, while Doc2a/Doc2bKO mice showed pronounced glucose intolerance. GSIS was markedly impaired in Doc2a/Doc2bKO mice in vivo, and in isolated Doc2a/Doc2bKO islets in vitro. In contrast, Doc2bKO mice had only subtle defects in insulin secretion in vivo. Insulin action was impaired to a similar degree in both Doc2bKO and Doc2a/Doc2bKO mice. In vitro insulin-stimulated glucose transport and GLUT4 vesicle fusion were defective in adipocytes derived from Doc2bKO mice. Surprisingly, insulin action was not altered in muscle isolated from DOC2-null mice. CONCLUSIONS/INTERPRETATION: Our study identifies a critical role for DOC2B in insulin-stimulated glucose uptake in adipocytes, and for the synergistic regulation of GSIS by DOC2A and DOC2B in beta cells.

Tomosyns attenuate SNARE assembly and synaptic depression by binding to VAMP2-containing template complexes.

Tomosyns are widely thought to attenuate membrane fusion by competing with synaptobrevin-2/VAMP2 for SNARE-complex assembly. Here, we present evidence against this scenario. In a novel mouse model, tomosyn-1/2 deficiency lowered the fusion barrier and enhanced the probability that synaptic vesicles fuse, resulting in stronger synapses with faster depression and slower recovery. While wild-type tomosyn-1m rescued these phenotypes, substitution of its SNARE motif with that of synaptobrevin-2/VAMP2 did not. Single-molecule force measurements indeed revealed that tomosyn's SNARE motif cannot substitute synaptobrevin-2/VAMP2 to form template complexes with Munc18-1 and syntaxin-1, an essential intermediate for SNARE assembly. Instead, tomosyns extensively bind synaptobrevin-2/VAMP2-containing template complexes and prevent SNAP-25 association. Structure-function analyses indicate that the C-terminal polybasic region contributes to tomosyn's inhibitory function. These results reveal that tomosyns regulate synaptic transmission by cooperating with synaptobrevin-2/VAMP2 to prevent SNAP-25 binding during SNARE assembly, thereby limiting initial synaptic strength and equalizing it during repetitive stimulation.

Tomosyn affects dense core vesicle composition but not exocytosis in mammalian neurons.

Tomosyn is a large, non-canonical SNARE protein proposed to act as an inhibitor of SNARE complex formation in the exocytosis of secretory vesicles. In the brain, tomosyn inhibits the fusion of synaptic vesicles (SVs), whereas its role in the fusion of neuropeptide-containing dense core vesicles (DCVs) is unknown. Here, we addressed this question using a new mouse model with a conditional deletion of tomosyn (Stxbp5) and its paralogue tomosyn-2 (Stxbp5l). We monitored DCV exocytosis at single vesicle resolution in tomosyn-deficient primary neurons using a validated pHluorin-based assay. Surprisingly, loss of tomosyns did not affect the number of DCV fusion events but resulted in a strong reduction of intracellular levels of DCV cargos, such as neuropeptide Y (NPY) and brain-derived neurotrophic factor (BDNF). BDNF levels were largely restored by re-expression of tomosyn but not by inhibition of lysosomal proteolysis. Tomosyn's SNARE domain was dispensable for the rescue. The size of the trans-Golgi network and DCVs was decreased, and the speed of DCV cargo flux through Golgi was increased in tomosyn-deficient neurons, suggesting a role for tomosyns in DCV biogenesis. Additionally, tomosyn-deficient neurons showed impaired mRNA expression of some DCV cargos, which was not restored by re-expression of tomosyn and was also observed in Cre-expressing wild-type neurons not carrying loxP sites, suggesting a direct effect of Cre recombinase on neuronal transcription. Taken together, our findings argue against an inhibitory role of tomosyns in neuronal DCV exocytosis and suggests an evolutionary conserved function of tomosyns in the packaging of secretory cargo at the Golgi.

DOC2B acts as a calcium switch and enhances vesicle fusion.

Calcium-dependent exocytosis is regulated by a vast number of proteins. DOC2B is a synaptic protein that translocates to the plasma membrane (PM) after small elevations in intracellular calcium concentration. The aim of this study was to investigate the role of DOC2B in calcium-triggered exocytosis. Using biochemical and biophysical measurements, we demonstrate that the C2A domain of DOC2B interacts directly with the PM in a calcium-dependent manner. Using a combination of electrophysiological, morphological, and total internal reflection fluorescent measurements, we found that DOC2B acts as a priming factor and increases the number of fusion-competent vesicles. Comparing secretion during repeated stimulation between wild-type DOC2B and a mutated DOC2B that is constantly at the PM showed that DOC2B enhances catecholamine secretion also during repeated stimulation and that DOC2B has to translocate to the PM to exert its facilitating effect, suggesting that its activity is dependent on calcium. The hypothesis that DOC2B exerts its effect at the PM was supported by the finding that DOC2B affects the fusion kinetics of single vesicles and interacts with the PM SNAREs (soluble NSF attachment receptors). We conclude that DOC2B is a calcium-dependent priming factor and its activity at the PM enables efficient expansion of the fusion pore, leading to increased catecholamine release.

Doc2b Ca2+ binding site mutants enhance synaptic release at rest at the expense of sustained synaptic strength.

Communication between neurons involves presynaptic neurotransmitter release which can be evoked by action potentials or occur spontaneously as a result of stochastic vesicle fusion. The Ca2+-binding double C2 proteins Doc2a and -b were implicated in spontaneous and asynchronous evoked release, but the mechanism remains unclear. Here, we compared wildtype Doc2b with two Ca2+ binding site mutants named DN and 6A, previously classified as gain- and loss-of-function mutants. They carry the substitutions D218,220N or D163,218,220,303,357,359A respectively. We found that both mutants bound phospholipids at low Ca2+ concentrations and were membrane-associated in resting neurons, thus mimicking a Ca2+-activated state. Their overexpression in hippocampal primary cultured neurons had similar effects on spontaneous and evoked release, inducing high mEPSC frequencies and increased short-term depression. Together, these data suggest that the DN and 6A mutants both act as gain-of-function mutants at resting conditions.

Transcription Factor 2I Regulates Neuronal Development via TRPC3 in 7q11.23 Disorder Models.

Williams syndrome (WS) and 7q11.23 duplication syndrome (Dup7q11.23) are neurodevelopmental disorders caused by the deletion and duplication, respectively, of ~ 25 protein-coding genes on chromosome 7q11.23. The general transcription factor 2I (GTF2I, protein TFII-I) is one of these proteins and has been implicated in the neurodevelopmental phenotypes of WS and Dup7q11.23. Here, we investigated the effect of copy number alterations in Gtf2i on neuronal maturation and intracellular calcium entry mechanisms known to be associated with this process. Mice with a single copy of Gtf2i (Gtf2i+/Del) had increased axonal outgrowth and increased TRPC3-mediated calcium entry upon carbachol stimulation. In contrast, mice with 3 copies of Gtf2i (Gtf2i+/Dup) had decreases in axon outgrowth and in TRPC3-mediated calcium entry. The underlying mechanism was that TFII-I did not affect TRPC3 protein expression, while it regulated TRPC3 membrane translocation. Together, our results provide novel functional insight into the cellular mechanisms that underlie neuronal maturation in the context of the 7q11.23 disorders.

SICT: automated detection and supervised inspection of fast Ca2+ transients.

Recent advances in live Ca2+ imaging with increasing spatial and temporal resolution offer unprecedented opportunities, but also generate an unmet need for data processing. Here we developed SICT, a MATLAB program that automatically identifies rapid Ca2+ rises in time-lapse movies with low signal-to-noise ratios, using fluorescent indicators. A graphical user interface allows visual inspection of automatically detected events, reducing manual labour to less than 10% while maintaining quality control. The detection performance was tested using synthetic data with various signal-to-noise ratios. The event inspection phase was evaluated by four human observers. Reliability of the method was demonstrated in a direct comparison between manual and SICT-aided analysis. As a test case in cultured neurons, SICT detected an increase in frequency and duration of spontaneous Ca2+ transients in the presence of caffeine. This new method speeds up the analysis of elementary Ca2+ transients.

Doc2B acts as a calcium sensor for vesicle priming requiring synaptotagmin-1, Munc13-2 and SNAREs.

Doc2B is a cytosolic protein with binding sites for Munc13 and Tctex-1 (dynein light chain), and two C2-domains that bind to phospholipids, Ca2+ and SNAREs. Whether Doc2B functions as a calcium sensor akin to synaptotagmins, or in other calcium-independent or calcium-dependent capacities is debated. We here show by mutation and overexpression that Doc2B plays distinct roles in two sequential priming steps in mouse adrenal chromaffin cells. Mutating Ca2+-coordinating aspartates in the C2A-domain localizes Doc2B permanently at the plasma membrane, and renders an upstream priming step Ca2+-independent, whereas a separate function in downstream priming depends on SNARE-binding, Ca2+-binding to the C2B-domain of Doc2B, interaction with ubMunc13-2 and the presence of synaptotagmin-1. Another function of Doc2B - inhibition of release during sustained calcium elevations - depends on an overlapping protein domain (the MID-domain), but is separate from its Ca2+-dependent priming function. We conclude that Doc2B acts as a vesicle priming protein.

Tomosyn associates with secretory vesicles in neurons through its N- and C-terminal domains.

The secretory pathway in neurons requires efficient targeting of cargos and regulatory proteins to their release sites. Tomosyn contributes to synapse function by regulating synaptic vesicle (SV) and dense-core vesicle (DCV) secretion. While there is large support for the presynaptic accumulation of tomosyn in fixed preparations, alternative subcellular locations have been suggested. Here we studied the dynamic distribution of tomosyn-1 (Stxbp5) and tomosyn-2 (Stxbp5l) in mouse hippocampal neurons and observed a mixed diffuse and punctate localization pattern of both isoforms. Tomosyn-1 accumulations were present in axons and dendrites. As expected, tomosyn-1 was expressed in about 75% of the presynaptic terminals. Interestingly, also bidirectional moving tomosyn-1 and -2 puncta were observed. Despite the lack of a membrane anchor these puncta co-migrated with synapsin and neuropeptide Y, markers for respectively SVs and DCVs. Genetic blockade of two known tomosyn interactions with synaptotagmin-1 and its cognate SNAREs did not abolish its vesicular co-migration, suggesting an interplay of protein interactions mediated by the WD40 and SNARE domains. We hypothesize that the vesicle-binding properties of tomosyns may control the delivery, pan-synaptic sharing and secretion of neuronal signaling molecules, exceeding its canonical role at the plasma membrane.

δ-Catenin (CTNND2) missense mutation in familial cortical myoclonic tremor and epilepsy.

OBJECTIVE: To identify the causative gene in a large Dutch family with familial cortical myoclonic tremor and epilepsy (FCMTE). METHODS: We performed exome sequencing for 3 patients of our FCMTE family. Next, we performed knock-down (shRNA) and rescue experiments by overexpressing wild-type and mutant human δ-catenin (CTNND2) proteins in cortical mouse neurons and compared the results with morphologic abnormalities in the postmortem FCMTE brain. RESULTS: We identified a missense mutation, p.Glu1044Lys, in the CTNND2 gene that cosegregated with the FCMTE phenotype. The knock-down of Ctnnd2 in cultured cortical mouse neurons revealed increased neurite outgrowth that was rescued by overexpression of wild-type, but not mutant, CTNND2 and was reminiscent of the morphologic abnormalities observed in cerebellar Purkinje cells from patients with FCMTE. CONCLUSIONS: We propose CTNND2 as the causal gene in FCMTE3. Functional testing of the mutant protein revealed abnormal neuronal sprouting, consistent with the abnormal cerebellar Purkinje cell morphology in patients with FCMTE.

Direct quantitative detection of Doc2b-induced hemifusion in optically trapped membranes.

Ca(2+)-sensor proteins control the secretion of many neuroendocrine substances. Calcium-secretion coupling may involve several mechanisms. First, Ca(2+)-dependent association of their tandem C2 domains with phosphatidylserine may induce membrane curvature and thereby enhance fusion. Second, their association with SNARE complexes may inhibit membrane fusion in the absence of a Ca(2+) trigger. Here we present a method using two optically trapped beads coated with SNARE-free synthetic membranes to elucidate the direct role of the C2AB domain of the soluble Ca(2+)-sensor Doc2b. Contacting membranes are often coupled by a Doc2b-coated membrane stalk that resists forces up to 600 pN upon bead separation. Stalk formation depends strictly on Ca(2+) and phosphatidylserine. Real-time fluorescence imaging shows phospholipid but not content mixing, indicating membrane hemifusion. Thus, Doc2b acts directly on membranes and stabilizes the hemifusion intermediate in this cell-free system. In living cells, this mechanism may co-occur with progressive SNARE complex assembly, together defining Ca(2+)-secretion coupling.

Tomosyn-2 is required for normal motor performance in mice and sustains neurotransmission at motor endplates.

Tomosyn-1 (STXBP5) is a soluble NSF attachment protein receptor complex-binding protein that inhibits vesicle fusion, but the role of tomosyn-2 (STXBP5L) in the mammalian nervous system is still unclear. Here we generated tomosyn-2 null (Tom2(KO/KO)) mice, which showed impaired motor performance. This was accompanied by synaptic changes at the neuromuscular junction, including enhanced spontaneous acetylcholine release frequency and faster depression of muscle motor endplate potentials during repetitive stimulation. The postsynaptic geometric arrangement and function of acetylcholine receptors were normal. We conclude that tomosyn-2 supports motor performance by regulation of transmitter release willingness to sustain synaptic strength during high-frequency transmission, which makes this gene a candidate for involvement in neuromuscular disorders.

Two male adults with pathogenic AUTS2 variants, including a two-base pair deletion, further delineate the AUTS2 syndrome.

AUTS2 syndrome is characterized by low birth weight, feeding difficulties, intellectual disability, microcephaly and mild dysmorphic features. All affected individuals thus far were caused by chromosomal rearrangements, variants at the base pair level disrupting AUTS2 have not yet been described. Here we present the full clinical description of two affected men with intragenic AUTS2 variants (one two-base pair deletion in exon 7 and one deletion of exon 6). Both variants are de novo and are predicted to cause a frameshift of the full-length transcript but are unlikely to affect the shorter 3' transcript starting in exon 9. The similarities between the phenotypes of both men are striking and further support that AUTS2 syndrome is a single gene disorder.

Identification of a Dutch founder mutation in MUSK causing fetal akinesia deformation sequence.

Fetal akinesia deformation sequence (FADS) refers to a clinically and genetically heterogeneous group of disorders with congenital malformations related to impaired fetal movement. FADS can result from mutations in CHRNG, CHRNA1, CHRND, DOK7 and RAPSN; however, these genes only account for a minority of cases. Here we identify MUSK as a novel cause of lethal FADS. Fourteen affected fetuses from a Dutch genetic isolate were traced back to common ancestors 11 generations ago. Homozygosity mapping in two fetuses revealed MUSK as a candidate gene. All tested cases carried an identical homozygous variant c.1724T>C; p.(Ile575Thr) in the intracellular domain of MUSK. The carrier frequency in the genetic isolate was 8%, exclusively found in heterozygous carriers. Consistent with the established role of MUSK as a tyrosine kinase that orchestrates neuromuscular synaptogenesis, the fetal myopathy was accompanied by impaired acetylcholine receptor clustering and reduced tyrosine kinase activity at motor nerve endings. A functional assay in myocytes derived from human fetuses confirmed that the variant blocks MUSK-dependent motor endplate formation. Taken together, the results strongly support a causal role of this founder mutation in MUSK, further expanding the gene set associated with FADS and offering new opportunities for prenatal genetic testing.

Tomosyn interacts with the SUMO E3 ligase PIASγ.

Protein modification by Small Ubiquitin-like MOdifier (SUMO) entities is involved in a number of neuronal functions, including synaptogenesis and synaptic plasticity. Tomosyn-1 (syntaxin-binding protein 5; STXPB5) binds to t-SNARE (Soluble NSF Attachment Protein Receptor) proteins to regulate neurotransmission and is one of the few neuronal SUMO substrate proteins identified. Here we used yeast two-hybrid screening to show that tomosyn-1 interacts with the SUMO E3 ligase PIASγ (Protein Inhibitor of Activated STAT; PIAS4 or ZMIZ6). This novel interaction involved the C-terminus of tomosyn-1 and the N-terminus of PIASγ. It was confirmed by two-way immunoprecipitation experiments using the full-length proteins expressed in HEK293T cells. Tomosyn-1 was preferentially modified by the SUMO-2/3 isoform. PIASγ-dependent modification of tomosyn-1 with SUMO-2/3 presents a novel mechanism to adapt secretory strength to the dynamic synaptic environment.

Genetic and phenotypic heterogeneity in sporadic and familial forms of paroxysmal dyskinesia.

Paroxysmal dyskinesia (PxD) is a group of movement disorders characterized by recurrent episodes of involuntary movements. Familial paroxysmal kinesigenic dyskinesia (PKD) is caused by PRRT2 mutations, but a distinct etiology has been suggested for sporadic PKD. Here we describe a cohort of patients collected from our movement disorders outpatient clinic in the period 1996-2011. Fifteen patients with sporadic PxD and 23 subjects from three pedigrees with familial PKD were screened for mutations in candidate genes. PRRT2 mutations co-segregated with PKD in two families and occurred in two sporadic cases of PKD. No mutations were detected in patients with non-kinesigenic or exertion-induced dyskinesia, and none in other candidate genes including PNKD1 (MR-1) and SLC2A1 (GLUT1). Thus, PRRT2 mutations also cause sporadic PKD as might be expected given the variable expressivity and reduced penetrance observed in familial PKD. Further genetic heterogeneity is suggested by the absence of candidate gene mutations in both sporadic and familial PKD suggesting a contribution of other genes or non-coding regions.