Epilepsy is a devastating and poorly understood disease. Mutations in a secreted neuronal protein, leucine-rich glioma inactivated 1 (LGI1), were reported in patients with an inherited form of human epilepsy, autosomal dominant partial epilepsy with auditory features (ADPEAF). Here, we report an essential role of LGI1 as an antiepileptogenic ligand. We find that loss of LGI1 in mice (LGI1 −/− ) causes lethal epilepsy, which is specifically rescued by the neuronal expression of LGI1 transgene, but not LGI3. Moreover, heterozygous mice for the LGI1 mutation (LGI1 +/− ) show lowered seizure thresholds. Extracellularly secreted LGI1 links two epilepsy-related receptors, ADAM22 and ADAM23, in the brain and organizes a transsynaptic protein complex that includes presynaptic potassium channels and postsynaptic AMPA receptor scaffolds. A lack of LGI1 disrupts this synaptic protein connection and selectively reduces AMPA receptor-mediated synaptic transmission in the hippocampus. Thus, LGI1 may serve as a major determinant of brain excitation, and the LGI1 gene-targeted mouse provides a good model for human epilepsy.A ffecting 1-2% of the population, epilepsy is one of the most common neurological disorders. Epilepsy is characterized by recurrent unprovoked seizures and is caused by disturbances in the delicate balance between excitation and inhibition in neural circuits (1, 2). Recent human genetic studies have established the channelopathy concept for idiopathic (inherited) epilepsies: Many of the genes whose mutations cause human epilepsy encode ion channel subunits (1, 2). Examples include voltagegated ion channels (K + , Na + , Ca 2+ , and Cl -channels) and ligandgated ion channels (nicotinic acetylcholine and GABA A receptors), which regulate neuronal excitability.Leucine-rich glioma inactivated 1 (LGI1) is a unique human epilepsy-related gene in that it does not encode an ion channel subunit (3-5), but is a neuronal secreted protein (6). Mutations in LGI1 are linked to autosomal dominant partial epilepsy with auditory features (ADPEAF, also known as autosomal dominant lateral temporal lobe epilepsy [ADLTE]) (3-5), which is an inherited epileptic syndrome characterized by partial seizures with acoustic or visual hallucinations. So far, 25 LGI1 mutations have been described in familial ADPEAF patients and sporadic cases (7). Interestingly, at least six tested ADPEAF mutations all abolish LGI1 secretion (6,8).Recent proteomic analysis identified LGI1 as a subunit of presynaptic Kv 1 (shaker type)-voltage gated potassium channels (9). It was shown that LGI1 selectively prevents inactivation of the Kv 1 channel mediated by a cytoplasmic regulatory protein, Kvβ. Because LGI1 is a secreted protein, it remains unclear how LGI1 modulates a cytosolic potassium channel mechanism. LGI1 was also isolated from the brain as a component of a protein complex mediated by PSD-95, a representative postsynaptic scaffolding protein.LGI1 functions as a ligand for the epilepsy-related ADAM22 transmembrane protein, which is anchored by PS...

Abnormally synchronized synaptic transmission in the brain causes epilepsy. Most inherited forms of epilepsy result from mutations in ion channels. However, one form of epilepsy, autosomal dominant partial epilepsy with auditory features (ADPEAF), is characterized by mutations in a secreted neuronal protein, LGI1. We show that ADAM22, a transmembrane protein that when mutated itself causes seizure, serves as a receptor for LGI1. LGI1 enhances AMPA receptor-mediated synaptic transmission in hippocampal slices. The mutated form of LGI1 fails to bind to ADAM22. ADAM22 is anchored to the postsynaptic density by cytoskeletal scaffolds containing stargazin. These studies in rat brain indicate possible avenues for understanding human epilepsy.

Protein palmitoylation is the most common posttranslational lipid modification; its reversibility mediates protein shuttling between intracellular compartments. A large family of DHHC (Asp-His-His-Cys) proteins has emerged as protein palmitoyl acyltransferases (PATs). However, mechanisms that regulate these PATs in a physiological context remain unknown. In this study, we efficiently monitored the dynamic palmitate cycling on synaptic scaffold PSD-95. We found that blocking synaptic activity rapidly induces PSD-95 palmitoylation and mediates synaptic clustering of PSD-95 and associated AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors. A dendritically localized DHHC2 but not the Golgi-resident DHHC3 mediates this activity-sensitive palmitoylation. Upon activity blockade, DHHC2 translocates to the postsynaptic density to transduce this effect. These data demonstrate that individual DHHC members are differentially regulated and that dynamic recruitment of protein palmitoylation machinery enables compartmentalized regulation of protein trafficking in response to extracellular signals.

The heterotrimeric G protein ␣ subunit (G␣) is targeted to the cytoplasmic face of the plasma membrane through reversible lipid palmitoylation and relays signals from G-protein-coupled receptors (GPCRs) to its effectors. By screening 23 DHHC motif (Asp-His-His-Cys) palmitoyl acyl-transferases, we identified DHHC3 and DHHC7 as G␣ palmitoylating enzymes. DHHC3 and DHHC7 robustly palmitoylated G␣ q , G␣ s , and G␣ i2 in HEK293T cells. Knockdown of DHHC3 and DHHC7 decreased G␣ q/11 palmitoylation and relocalized it from the plasma membrane into the cytoplasm. Photoconversion analysis revealed that G␣ q rapidly shuttles between the plasma membrane and the Golgi apparatus, where DHHC3 specifically localizes. Fluorescence recovery after photobleaching studies showed that DHHC3 and DHHC7 are necessary for this continuous G␣ q shuttling. Furthermore, DHHC3 and DHHC7 knockdown blocked the ␣ 1A -adrenergic receptor/G␣ q/11 -mediated signaling pathway. Together, our findings revealed that DHHC3 and DHHC7 regulate GPCR-mediated signal transduction by controlling G␣ localization to the plasma membrane.G-protein-coupled receptors (GPCRs) form the largest family of cell surface receptors, consisting of more than 700 members in humans. GPCRs respond to a variety of extracellular signals, including hormones and neurotransmitters, and are involved in various physiologic processes, such as smooth muscle contraction and synaptic transmission (20,25). Heterotrimeric G proteins, composed of ␣, ␤, and ␥ subunits, transduce signals from GPCRs to their effectors and play a central role in the GPCR signaling pathway (13,21,24,32). Although the G␣ subunit seems to localize stably at the cytosolic face of the plasma membrane (PM), a recent report suggested that G␣ o , a G␣ isoform, shuttles rapidly between the PM and intracellular membranes (2). The PM targeting of G␣ requires both interaction with the G␤␥ complex and subsequent lipid palmitoylation of G␣ (22). Thus, palmitoylation of G␣ is a critical determinant of membrane targeting of the heterotrimer G␣␤␥.Protein palmitoylation is a common posttranslational modification with lipid palmitate and regulates protein trafficking and function (7,18). G␣ is a classic and representative palmitoyl substrate (19,38), and recent studies revealed that protein palmitoylation modifies virtually almost all the components of G-protein signaling, including GPCRs, G␣ subunits, several members of the RGS (regulators of G-protein signaling) family of GTPase-activating proteins, GPCR kinase GRK6, and some small GTPases (7, 33). This common lipid modification plays an important role in compartmentalizing G-protein signaling to the specific microdomain, such as membrane caveolae and lipid raft (26). The palmitoyl thioester bond is relatively labile, and palmitates on substrates turn over rapidly, allowing proteins to shuttle between the cytoplasm/intracellular organelles and the PM (2, 3, 27). For example, binding of isoproterenol to the ␤-adrenergic receptor markedly accelerates the depalmitoylation of the a...

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