Margaret H. Butler

Epsin is an EH-domain-binding protein implicated in clathrin-mediated endocytosis

During endocytosis, clathrin and the clathrin adaptor protein AP-2 (ref. 1), assisted by a variety of accessory factors, help to generate an invaginated bud at the cell membrane,. One of these factors is Eps15, a clathrin-coat-associated protein that binds the α-adaptin subunit of AP-2 (refs 4–8). Here we investigate the function of Eps15 by characterizing an important binding partner for its region containing EH domains; this protein, epsin, is closely related to the Xenopus mitotic phosphoprotein MP90 (ref. 10) and has a ubiquitous tissue distribution. It is concentrated together with Eps15 in presynaptic nerve terminals, which are sites specialized for the clathrin-mediated endocytosis of synaptic vesicles. The central region of epsin binds AP-2 and its carboxy-terminal region binds Eps15. Epsin is associated with clathrin coats in situ, can be co-precipitated with AP-2 and Eps15 from brain extracts, but does not co-purify with clathrin coat components in a clathrin-coated vesicle fraction. When epsin function is disrupted, clathrin-mediated endocytosis is blocked. We propose that epsin may participate, together with Eps15, in the molecular rearrangement of the clathrin coats that are required for coated-pit invagination and vesicle fission.

MiRP2 Forms Potassium Channels in Skeletal Muscle with Kv3.4 and Is Associated with Periodic Paralysis

The subthreshold, voltage-gated potassium channel of skeletal muscle is shown to contain MinK-related peptide 2 (MiRP2) and the pore-forming subunit Kv3.4. MiRP2-Kv3.4 channels differ from Kv3.4 channels in unitary conductance, voltage-dependent activation, recovery from inactivation, steady-state open probability, and block by a peptide toxin. Thus, MiRP2-Kv3.4 channels set resting membrane potential (RMP) and do not produce afterhyperpolarization or cumulative inactivation to limit action potential frequency. A missense mutation is identified in the gene for MiRP2 (KCNE3) in two families with periodic paralysis and found to segregate with the disease. Mutant MiRP2-Kv3.4 complexes exhibit reduced current density and diminished capacity to set RMP. Thus, MiRP2 operates with a classical potassium channel subunit to govern skeletal muscle function and pathophysiology.

Sumoylation Silences the Plasma Membrane Leak K+ Channel K2P1

Reversible, covalent modification with small ubiquitin-related modifier proteins (SUMOs) is known to mediate nuclear import/export and activity of transcription factors. Here, the SUMO pathway is shown to operate at the plasma membrane to control ion channel function. SUMO-conjugating enzyme is seen to be resident in plasma membrane, to assemble with K2P1, and to modify K2P1 lysine 274. K2P1 had not previously shown function despite mRNA expression in heart, brain, and kidney and sequence features like other two-P loop K+ leak (K2P) pores that control activity of excitable cells. Removal of the peptide adduct by SUMO protease reveals K2P1 to be a K+-selective, pH-sensitive, openly rectifying channel regulated by reversible peptide linkage.

Autoimmunity to Gephyrin in Stiff-Man Syndrome

Stiff-Man syndrome (SMS) is a rare disease of the central nervous system (CNS) characterized by chronic rigidity, spasms, and autoimmunity directed against synaptic antigens, most often the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD). In a subset of cases, SMS has an autoimmune paraneoplastic origin. We report here the identification of high-titer autoantibodies directed against gephyrin in a patient with clinical features of SMS and mediastinal cancer. Gephyrin is a cytosolic protein selectively concentrated at the postsynaptic membrane of inhibitory synapses, where it is associated with GABA(A) and glycine receptors. Our findings provide new evidence for a close link between autoimmunity directed against components of inhibitory synapses and neurological conditions characterized by chronic rigidity and spasms.

SYNAPTOJANIN 1 : LOCALIZATION ON COATED ENDOCYTIC INTERMEDIATES IN NERVE TERMINALS AND INTERACTION OF ITS 170 KDA ISOFORM WITH EPS15

Synaptojanin 1 is an inositol 5‐phosphatase with a putative role in clathrin‐mediated endocytosis. Goal of this study was to provide new evidence for this hypothesis. We show that synaptojanin 1 is concentrated at clathrin‐coated endocytic intermediates in nerve terminals. Furthermore, we report that synaptojanin‐170, an alternatively spliced isoform of synaptojanin 1, binds Eps15, a clathrin coat‐associated protein. Binding is mediated by the COOH‐terminal region of synaptojanin‐170 which we show here to be poorly conserved from rat to humans, but to contain in both species three asparagine‐proline‐phenylalanine (NPF) repeats. This motif has been found to be the core of the binding site for the EH domains of Eps15. Together with previous data, our results suggest that synaptojanin 1 can be recruited to clathrin‐coated pits via a multiplicity of interactions.

Tandem Arrangement of the Clathrin and AP-2 Binding Domains in Amphiphysin 1 and Disruption of Clathrin Coat Function by Amphiphysin Fragments Comprising These Sites

Amphiphysin 1 and 2 are proteins implicated in the recycling of synaptic vesicles in nerve terminals. They interact with dynamin and synaptojanin via their COOH-terminal SH3 domain, whereas their central regions contain binding sites for clathrin and for the clathrin adaptor AP-2. We have defined here amino acids of amphiphysin 1 crucial for binding to AP-2 and clathrin. Overexpression in Chinese hamster ovary cells of an amphiphysin 1 fragment that binds both AP-2 and clathrin resulted in a segregation of clathrin, which acquired a diffuse distribution, from AP-2, which accumulated at patches also positive for Eps15. These effects correlated with a block in clathrin-mediated endocytosis. A fragment selectively interacting with clathrin produced a similar effect. These results can be explained by the binding of amphiphysin to the NH2-terminal domain of clathrin and by a competition with the binding of this domain to the β-subunit of AP-2 and AP180. The interaction of amphiphysin 1 with either clathrin or AP-2 did not prevent its interaction with dynamin, supporting the existence of tertiary complexes between these proteins. Together with previous evidence indicating a direct interaction between amphiphysin and membrane lipids, these findings support a model in which amphiphysin acts as a multifunctional adaptor linking the membrane to coat proteins and coat proteins to dynamin and synaptojanin.

Anti-amphiphysin I antibodies in patients with paraneoplastic neurological disorders associated with small cell lung carcinoma.

Patients with stiff man syndrome and breast cancer develop anti-amphiphysin I antibodies that primarily recognise the C terminus of the protein. Anti-amphiphysin I antibodies have also been identified in a few patients with paraneoplastic neurological disorders (PND) and small cell lung cancer (SCLC). The frequency of anti-amphiphysin I antibodies in patients with SCLC and PND was analysed and the epitope specificity of these antibodies was characterised.  Anti-amphiphysin I antibodies were evaluated by immunohistochemistry on human and rat cerebellum and immunoblots of rat brain homogenates. Serum samples included 134 patients with PND and anti-Hu antibodies (83% had SCLC), 44 with SCLC and PND without antiHu-antibodies, 63 with PND and either Yo, Ri, or Tr antibodies, 146 with SCLC without PND, and 104 with non-PND. Positive serum samples were confirmed with immunoblots of recombinant human amphiphysin I and immunoreacted with five overlapping peptide fragments covering the full length of the molecule.  Serum samples positive for anti-amphiphysin I antibodies included those from seven (2.9%) patients with PND and two (1.4%) with SCLC without PND. Six of the seven anti-amphiphysin I antibody positive patients with PND had SCLC (three with Hu-antibodies), and one had anti-Hu-antibodies but no detectable tumour. The PND included encephalomyelitis/sensory neuropathy (five patients), cerebellar degeneration (one), and opsoclonus (one). All anti-amphiphysin I antibodies reacted with the C terminus of amphiphysin I, but seven also recognised other fragments of the molecule.  In conclusion, anti-amphiphysin I antibodies are present at low frequency in patients with SCLC irrespective of the presence of an associated PND. All anti-amphiphysin I antibody positive serum samples have in common reactivity with the C terminus of the protein.

Ito Channels Are Octomeric Complexes with Four Subunits of Each Kv4.2 and K+ Channel-interacting Protein 2

Mammalian voltage-gated K+ channels are assemblies of pore-forming α-subunits and modulating β-subunits. To operate correctly, Kv4 α-subunits in the heart and central nervous system require recently identified β-subunits of the neuronal calcium sensing protein family called K+ channel-interacting proteins (KChIPs). Here, Kv4.2·KChIP2 channels are purified, integrity of isolated complexes confirmed, molar ratio of the subunits determined, and subunit valence established. A complex has 4 subunits of each type, a stoichiometry expected for other channels employing neuronal calcium sensing β-subunits.

A novel antineuronal antibody in a motor neuron syndrome associated with breast cancer

Article abstract A 72-year-old woman developed a lower motor neuron syndrome (MNS) 4 months before the appearance of breast cancer. Monoparesis progressed to quadriparesis despite high-dose IV immunoglobulins, plasma exchange, and azathioprine, and high-dose IV methylprednisolone. The patient improved only after the removal of the tumor. MRI demonstrated hyperintensities in the cervical spinal cord. The patient had antibodies that reacted with axonal initial segments and nodes of Ranvier. The findings suggest that in this patient lower MNS may be a paraneoplastic condition associated with breast cancer.

Autoimmunity to βIV spectrin in paraneoplastic lower motor neuron syndrome

Paraneoplastic neurological disorders may result from autoimmunity directed against antigens shared by the affected neurons and the associated cancer cells. We have recently reported the case of a woman with breast cancer and paraneoplastic lower motor neuron syndrome whose serum contained autoantibodies directed against axon initial segments and nodes of Ranvier of myelinated axons, including the axons of motoneurons. Here, we show that major targets of the autoantibodies of this patient are βIVΣ1 spectrin and βIV spectrin 140, two isoforms of the novel βIV spectrin gene, as well as a neuronal surface epitope yet to be identified. Partial improvement of the neurological symptoms following cancer removal was associated with a drastic reduction in the titer of the autoantibodies against βIV spectrin and nodal antigens in general, consistent with the autoimmune pathogenesis of the paraneoplastic lower motor neuron syndrome. The identification of βIV spectrin isoforms and surface nodal antigens as novel autoimmune targets in lower motor neuron syndrome provide new insights into the pathogenesis of this severe neurological disease.