Generalized epilepsy with febrile seizures plus type 1 is an inherited human epileptic syndrome, associated with a cysteine-to-tryptophan (C121W) mutation in the extracellular immunoglobin domain of the auxiliary beta1 subunit of the voltage-gated sodium channel. The mutation disrupts beta1 function, but how this leads to epilepsy is not understood. In this study, we make several observations that may be relevant for understanding why this beta1 mutation results in seizures. First, using electrophysiological recordings from mammalian cell lines, coexpressing sodium channel alpha subunits and either wild-type beta1 or C121Wbeta1, we show that loss of beta1 functional modulation, caused by the C121W mutation, leads to increased sodium channel availability at hyperpolarized membrane potentials and reduced sodium channel rundown during high-frequency channel activity, compared with channels coexpressed with wild-type beta1. In contrast, neither wild-type beta1 nor C121Wbeta1 significantly affected sodium current time course or the voltage dependence of channel activation. We also show, using a Drosophila S2 cell adhesion assay, that the C121W mutation disrupts beta1-beta1 homophilic cell adhesion, suggesting that the mutation may alter the ability of beta1 to mediate protein-protein interactions critical for sodium channel localization. Finally, we demonstrate that neither functional modulation nor cell adhesion mediated by wild-type beta1 is occluded by coexpression of C121Wbeta1, arguing against the idea that the mutant beta1 acts as a dominant-negative subunit. Together, these data suggest that C121Wbeta1 causes subtle effects on channel function and subcellular distribution that bias neurons toward hyperexcitabity and epileptogenesis.

Voltage-gated sodium channel ␣ and ␤ subunits expressed in mammalian heart are differentially localized to t-tubules and intercalated disks. Sodium channel ␤ subunits are multifunctional molecules that participate in channel modulation and cell adhesion. Reversible, receptor-mediated changes in ␤1 tyrosine phosphorylation modulate its ability to recruit and associate with ankyrin. The purpose of the present study was to test our hypothesis that tyrosine-phosphorylated ␤1 (pY␤1) and nonphosphorylated ␤1 subunits may be differentially localized in heart and thus interact with different cytoskeletal and signaling proteins. We developed an antibody that specifically recognizes pY␤1 and investigated the differential subcellular localization of ␤1 and pY␤1 in mouse ventricular myocytes. We found that pY␤1 colocalized with connexin-43, N-cadherin, and Na v 1.5 at intercalated disks but was not detected at the t-tubules. Anti-pY␤1 immunoprecipitates N-cadherin from heart membranes and from cells transfected with ␤1 and N-cadherin in the absence of other sodium channel subunits. pY␤1 does not associate with ankyrin B in heart membranes. N-cadherin and connexin-43 associate with Na v 1.5 in heart membranes as assessed by co-immunoprecipitation assays. We propose that sodium channel complexes at intercalated disks of ventricular myocytes are composed of Na v 1.5 and pY␤1 and that these complexes are in close association with both N-cadherin and connexin-43. ␤1 phosphorylation appears to regulate its localization to differential subcellular domains.

Voltage-gated sodium channels are composed of a pore-forming ␣ subunit and at least one auxiliary ␤ subunit. Both ␤1 and ␤2 are cell adhesion molecules that interact homophilically, resulting in ankyrin recruitment. In contrast, ␤1, but not ␤2, interacts heterophilically with contactin, resulting in increased levels of cell surface sodium channels. We took advantage of these results to investigate the molecular basis of ␤1-mediated enhancement of sodium channel cell surface density, including elucidating structure-function relationships for ␤1 association with contactin, ankyrin, and Na v 1.2. ␤1/␤2 subunit chimeras were used to assign putative sites of contactin interaction to two regions of the ␤1 Ig loop. Recent studies have shown that glutathione S-transferase fusion proteins containing portions of Na v 1.2 intracellular domains interact directly with ankyrin G . We show that native Na v 1.2 associates with ankyrin G in cells in the absence of ␤ subunits and that this interaction is enhanced in the presence of ␤1 but not ␤1Y181E, a mutant that does not interact with ankyrin G . ␤1Y181E does not modulate Na v 1.2 channel function despite efficient association with Na v 1.2 and contactin. ␤1Y181E increases Na v 1.2 cell surface expression, but not as efficiently as wild type ␤1. ␤1/␤2 chimeras exchanging various regions of the ␤1 Ig loop were all ineffective in increasing Na v 1.2 cell surface density. Our results demonstrate that full-length ␤1 is required for channel modulation and enhancement of sodium channel cell surface expression.Voltage-gated sodium channels consist of a pore-forming ␣ subunit and at least one ␤ subunit (1-4). ␤1, ␤1A, and ␤3 non-covalently associate with ␣, whereas ␤2 and ␤4 are disulfide-linked to ␣ (4 -7). ␤ subunits have been shown to modulate channel kinetics and regulate cell surface expression of ␣ subunits (5, 8).Sodium channel ␤ subunits are unique among voltage-gated ion channel auxiliary subunits in that they also function as cell adhesion molecules (9). ␤1 and ␤2 interact with tenascin-C and tenascin-R, resulting in cellular repulsion (10, 11), and participate in homophilic (␤1-␤1 or ␤2-␤2) cell adhesion resulting in ankyrin recruitment to the plasma membrane at points of cell-cell contact (12). Ankyrin recruitment by ␤1 is dependent on phosphorylation of an intracellular tyrosine residue, ␤1Y181. Phosphorylation of ␤1Y181, or mutation of the tyrosine to glutamate mimicking a phosphorylation state (␤1Y181E), inhibits ␤1-mediated ankyrin recruitment (13) and results in ␤1 targeting to ankyrin-independent subcellular domains (14). ␤ subunits also participate in heterophilic celladhesive interactions with other Ig superfamily cell adhesion molecules. ␤1 interacts with contactin in vitro, resulting in increased Na v 1.2 cell surface expression without affecting channel function (15). Both ␤1 and ␤3 interact with neurofascin-186 (16), and the intracellular domain of ␤1 has been shown to interact with the receptor protein-tyrosine phosphatase ␤ (17).The purpose of the present stu...

Single-center studies have shown acceptable long-term outcomes following orthotopic liver transplantation (OLT) for hepatocellular carcinoma (HCC) when tumors are within the Milan criteria. However, the overall survival and waiting list removal rates have not been described at a national level with pooled registry data. To evaluate this, a retrospective cohort of patients listed for OLT with a diagnosis of HCC between January 1998 and March 2006 was identified from Organ Procurement Transplant Network data. Analysis was performed from the time of listing. Adjusted Cox models were used to assess the relative effect of potential confounders on removal from the waiting list as well as survival from the time of wait listing. A total of 4482 patients with HCC were placed on the liver waiting list during the study period. Of these, 65% underwent transplantation, and 18% were removed from the list because of tumor progression or death. The overall 1-and 5-year intent-to-treat survival for all patients listed was 81% and 51%, respectively. The 1-and 5-year survival was 89% and 61% for those listed with tumors meeting the Milan criteria versus 70% and 32% for those exceeding the Milan criteria (P Ͻ 0.0001). On multivariate analysis, advanced liver failure manifested by Child-Pugh class B or C increased the risk of death, while age Ͻ 55 years, meeting the Milan criteria, and obtaining a liver transplant were associated with better survival. The current criteria for liver transplantation of candidates with HCC lead to acceptable 5-year survival while limiting the dropout rate. Liver Transpl 15:859-868, 2009.

This systematic review supports the extrapolation of efficacy results in adults to predict a similar adjunctive treatment response in 2- to 18-year-old children with POS.