Fast inactivation of voltage-gated sodium (Nav) channels is essential for electrical signaling, but its mechanism remains poorly understood. Here we determined the structures of a eukaryotic Nav channel alone and in complex with a lethal α-scorpion toxin, AaH2, by electron microscopy, both at 3.5-angstrom resolution. AaH2 wedges into voltage-sensing domain IV (VSD4) to impede fast activation by trapping a deactivated state in which gating charge interactions bridge to the acidic intracellular carboxyl-terminal domain. In the absence of AaH2, the S4 helix of VSD4 undergoes a ~13-angstrom translation to unlatch the intracellular fast-inactivation gating machinery. Highlighting the polypharmacology of α-scorpion toxins, AaH2 also targets an unanticipated receptor site on VSD1 and a pore glycan adjacent to VSD4. Overall, this work provides key insights into fast inactivation, electromechanical coupling, and pathogenic mutations in Nav channels.

Epidermal growth factor receptor (EGFR) signaling is a potent driver of glioblastoma, a malignant and lethal form of brain cancer. Disappointingly, inhibitors targeting receptor tyrosine kinase activity are not clinically effective, and EGFR persists on the plasma membrane to maintain tumor growth and invasiveness. Here we show that endolysosomal pH is critical for receptor sorting and turnover. By functioning as a leak pathway for protons, the Na+/H+ exchanger NHE9 limits luminal acidification to circumvent EGFR turnover and prolong downstream signaling pathways that drive tumor growth and migration. In glioblastoma, NHE9 expression is associated with stem/progenitor characteristics, radiochemoresistance, poor prognosis and invasive growth in vitro and in vivo. Silencing or inhibition of NHE9 in brain tumor initiating cells attenuates tumorsphere formation and improves efficacy of EGFR inhibitor. Thus, NHE9 mediates inside-out control of oncogenic signaling and is a highly druggable target for pan-specific receptor clearance in cancer therapy.

Progression of benign tumors to invasive, metastatic cancer is accompanied by the epithelial-to-mesenchymal transition (EMT), characterized by loss of the cell-adhesion protein E-cadherin. Although silencing mutations and transcriptional repression of the E-cadherin gene have been widely studied, not much is known about posttranslational regulation of E-cadherin in tumors. We show that E-cadherin is tightly coexpressed with the secretory pathway Ca 2þ -ATPase isoform 2, SPCA2 (ATP2C2), in breast tumors. Loss of SPCA2 impairs surface expression of E-cadherin and elicits mesenchymal gene expression through disruption of cell adhesion in tumorspheres and downstream Hippo-YAP signaling. Conversely, ectopic expression of SPCA2 in triple-negative breast cancer elevates baseline Ca 2þ and YAP phosphorylation, enhances posttranslational expression of E-cadherin, and suppresses mesenchymal gene expression. Thus, loss of SPCA2 phenocopies loss of E-cadherin in the Hippo signaling pathway and EMT-MET transitions, consistent with a functional role for SPCA2 in E-cadherin biogenesis. Furthermore, we show that SPCA2 suppresses invasive phenotypes, including cell migration in vitro and tumor metastasis in vivo. Based on these findings, we propose that SPCA2 functions as a key regulator of EMT and may be a potential therapeutic target for treatment of metastatic cancer.Implications: Posttranslational control of E-cadherin and the Hippo pathway by calcium signaling regulates EMT in breast cancer cells.

SummaryProgression of benign tumors to invasive, metastatic cancer requires loss of the celladhesion protein E-cadherin. Although intensive efforts have focused on gene repression and silencing mutations, much less is known about posttranslational control of E-cadherin expression in cancer. SPCA2 is a secretory pathway Ca 2+ -ATPase that is down-regulated in metastatic breast cancer. We show that SPCA2 is tightly co-expressed with epithelial signature genes and required for E-cadherin biogenesis and cell surface expression.Unexpectedly, this function is uncoupled from Ca 2+ pumping and mediated by binding to E-cadherin. Loss of SPCA2 is sufficient to disrupt cell-cell adhesion in tumorspheres and elicit mesenchymal gene expression through Hippo-YAP signaling. These findings point to a causal link between low SPCA2 levels and the epithelial-mesenchymal transition required for breast cancer metastasis.

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A comparative performance evaluation of DNA extraction methods from anti-diabetic botanical supplements using various commercial kits was conducted, to determine which produces the best quality DNA suitable for PCR amplification, sequencing and species identification. All plant materials involved were of suboptimal quality showing various levels of degradation and therefore representing real conditions for testing herbal supplements. Eight different DNA extraction methods were used to isolate genomic DNA from 13 medicinal plant products. Two methods for evaluation, DNA concentration measurements that included absorbance ratios as well as PCR amplifiability, were used to determine quantity and quality of extracted DNA. We found that neither DNA concentrations nor commonly used UV absorbance ratio measurements at A(260)/A(280) between 1.7 and 1.9 are suitable for globally predicting PCR success in these plant samples, and that PCR amplifiablity itself was the best indicator of extracted product quality. However, our results suggest that A(260)/A(280) ratios below about 1.3 and above 2.3 indicated a DNA quality too poor to amplify. Therefore, A(260)/A(280) measurements are not useful to identify samples that likely will amplify but can be used to exclude samples that likely will not amplify reducing the cost for unnecessarily subjecting samples to PCR. The two Nucleospin(®) plant II kit extraction methods produced the most pure and amplifiable genomic DNA extracts. Our results suggest that there are clear, discernable differences between extraction methods for low quality plant samples in terms of producing contamination-free, high-quality genomic DNA to be used for further analysis.

Background: Voltage-gated sodium (Na V) channels help regulate electrical activity of the plasma membrane. Mutations in associated subunits can result in pathological outcomes. Here we examined the interaction of Na V channels with cardiac arrhythmia-linked mutations in SCN2B and SCN4B, two genes that encode auxiliary b-subunits. Materials and Methods: To investigate changes in SCN2B R137H and SCN4B I80T function, we combined threedimensional X-ray crystallography with electrophysiological measurements on Na V 1.5, the dominant subtype in the heart. Results: SCN4B I80T alters channel activity, whereas SCN2B R137H does not have an apparent effect. Structurally, the SCN4B I80T perturbation alters hydrophobic packing of the subunit with major structural changes and causes a thermal destabilization of the folding. In contrast, SCN2B R137H leads to structural changes but overall protein stability is unaffected. Conclusion: SCN4B I80T data suggest a functionally important region in the interaction between Na V 1.5 and b4 that, when disrupted, could lead to channel dysfunction. A lack of apparent functional effects of SCN2B R137H on Na V 1.5 suggests an alternative working mechanism, possibly through other Na V channel subtypes present in heart tissue. Indeed, mapping the structural variations of SCN2B R137H onto neuronal Na V channel structures suggests altered interaction patterns.

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