Spinocerebellar ataxia type 13 mutant potassium channel alters neuronal excitability and causes locomotor deficits in zebrafish.

Whether changes in neuronal excitability can cause neurodegenerative disease in the absence of other factors such as protein aggregation is unknown. Mutations in the Kv3.3 voltage-gated K(+) channel cause spinocerebellar ataxia type 13 (SCA13), a human autosomal-dominant disease characterized by locomotor impairment and the death of cerebellar neurons. Kv3.3 channels facilitate repetitive, high-frequency firing of action potentials, suggesting that pathogenesis in SCA13 is triggered by changes in electrical activity in neurons. To investigate whether SCA13 mutations alter excitability in vivo, we expressed the human dominant-negative R420H mutant subunit in zebrafish. The disease-causing mutation specifically suppressed the excitability of Kv3.3-expressing, fast-spiking motor neurons during evoked firing and fictive swimming and, in parallel, decreased the precision and amplitude of the startle response. The dominant-negative effect of the mutant subunit on K(+) current amplitude was directly responsible for the reduced excitability and locomotor phenotype. Our data provide strong evidence that changes in excitability initiate pathogenesis in SCA13 and establish zebrafish as an excellent model system for investigating how changes in neuronal activity impair locomotor control and cause cell death.

Spontaneous autoinflation of saline mammary implants: further studies.

BACKGROUND: Previous studies have reported a hyperinflation of saline-filled breast implants. On removal, the implant fluid had changed from clear saline to a yellowish-brown color, with a viscous consistency similar to serum. OBJECTIVE: Our objective was to identify further the components of saline from implants that had undergone spontaneous autoinflation. Our hypothesis was that if serum albumin is present in the fluid, then other proteins would likely be found. METHODS: To screen and identify proteins in implant fluid, we used a proteomics-based approach that included 1- and 2-dimensional gel electrophoresis and mass spectrometry of protein samples. RESULTS: Four known proteins and 1 unknown protein product were identified. Based on 2-dimensional gel electrophoresis and mass spectrometry, 2 general observations can be made about the saline from the autoinflated implants: serum albumin was the most prevalent protein, and there are a large number of proteins that remain to be identified. CONCLUSIONS: There are multiple macromolecules that cross into the lumen of the prosthesis. We believe spontaneous autoinflation is occurring more often than is believed or reported.

The carboxyl terminus of the alpha-subunit of the amiloride-sensitive epithelial sodium channel binds to F-actin.

The activity of the amiloride-sensitive epithelial sodium channel (ENaC) is modulated by F-actin. However, it is unknown if there is a direct interaction between alpha-ENaC and actin. We have investigated the hypothesis that the actin cytoskeleton directly binds to the carboxyl terminus of alpha-ENaC using a combination of confocal microscopy, co-immunoprecipitation, and protein binding studies. Confocal microscopy of Madin-Darby canine kidney cell monolayers stably transfected with wild type, rat isoforms of alpha-, beta-, and gamma-ENaC revealed co-localization of alpha-ENaC with the cortical F-actin cytoskeleton both at the apical membrane and within the subapical cytoplasm. F-actin was found to co-immunoprecipitate with alpha-ENaC from whole cell lysates of this cell line. Gel overlay assays demonstrated that F-actin specifically binds to the carboxyl terminus of alpha-ENaC. A direct interaction between F-actin and the COOH terminus of alpha-ENaC was further corroborated by F-actin co-sedimentation studies. This is the first study to report a direct and specific biochemical interaction between F-actin and ENaC.