Abstract-Brugada syndrome is an inherited cardiac disorder caused by mutations in the cardiac sodium channel gene, SCN5A, that leads to ventricular fibrillation and sudden death. This study reports the changes in functional expression and cellular localization of an SCN5A double mutant (R1232W/T1620M) recently discovered in patients with Brugada syndrome. Mutant and wild-type (WT) human heart sodium channels (hNa v 1.5) were expressed in tsA201 cells in the presence of the ␤ 1 -auxiliary subunit. Patch-clamp experiments in whole-cell configuration were conducted to assess functional expression. Immunohistochemistry and confocal microscopy were used to determine the spatial distribution of either WT or mutant cardiac sodium channels. The results show an abolition of functional sodium channel expression of the hNa v 1.5/R1232W/T1620M mutant in the tsA201 cells. A conservative positively charged mutant, hNa v 1.5/ R1232K/T1620M, produced functional channels. Immunofluorescent staining showed that the FLAG-tagged hNa v 1.5/WT transfected into tsA201 cells was localized on the cell surface, whereas the FLAG-tagged hNa v 1.5/ R1232W/T1620M mutant was colocalized with calnexin within the endoplasmic reticulum (ER

No abstract

No abstract

We previously described the isolation of yeast mutants (sex mutants) that secrete reduced amounts of mature alpha-factor when it is synthesized as part of a fusion with prosomatostatin. In the present study we show that the sex3-1 mutant displays pleiotropic phenotypes. These include an abnormal morphology, an osmoremediable caffeine sensitivity, reduced secretion of mature alpha-factor, a weakened cell wall and a marked deficiency in halotolerance. Cloning of the SEX3 gene revealed that it is identical to the RPB4 gene. This gene encodes the fourth largest subunit of yeast RNA polymerase II, which has been postulated to play a major role in the response to stress. We show that transcriptional activation in response to either a cell wall stress or to growth in the presence of elevated salt concentrations is minimally affected by the loss of RPB4 function. However, whereas the levels of several mRNAs are similarly reduced (by about 30%) in rpb4 mutants grown in rich medium at moderate temperature, some transcripts, in particular ZDS1, are more abundant. An increase dosage of ZDS1, or of genes involved in cell wall assembly and in secretion (RHO1 and SR077, respectively), partially suppresses the sensitivity of rpb4delta cells to high temperature, heat shock and stationary phase. Collectively, our results indicate that the loss of Rpb4p perturbs several cellular functions that contribute to the inappropriate stress response of rpb4delta yeast. We therefore conclude that this RNA poiymerase II subunit is not specifically involved in the stress response.