Oxidative protein folding in the endoplasmic reticulum (ER) has emerged as a potentially significant source of cellular reactive oxygen species (ROS). Recent studies suggest that levels of ROS generated as a byproduct of oxidative folding rival those produced by mitochondrial respiration. Mechanisms that protect cells against oxidant accumulation within the ER have begun to be elucidated yet many questions still remain regarding how cells prevent oxidant-induced damage from ER folding events. Here we report a new role for a central well-characterized player in ER homeostasis as a direct sensor of ER redox imbalance. Specifically we show that a conserved cysteine in the lumenal chaperone BiP is susceptible to oxidation by peroxide, and we demonstrate that oxidation of this conserved cysteine disrupts BiP's ATPase cycle. We propose that alteration of BiP activity upon oxidation helps cells cope with disruption to oxidative folding within the ER during oxidative stress.DOI: http://dx.doi.org/10.7554/eLife.03496.001

Unfavorable redox conditions in the endoplasmic reticulum (ER) can decrease the capacity for protein secretion, altering vital cell functions. While systems to manage reductive stress are well-established, how cells cope with an overly oxidizing ER remains largely undefined. In previous work (Wang et al., 2014), we demonstrated that the chaperone BiP is a sensor of overly oxidizing ER conditions. We showed that modification of a conserved BiP cysteine during stress beneficially alters BiP chaperone activity to cope with suboptimal folding conditions. How this cysteine is reduced to reestablish 'normal' BiP activity post-oxidative stress has remained unknown. Here we demonstrate that BiP's nucleotide exchange factor – Sil1 – can reverse BiP cysteine oxidation. This previously unexpected reductant capacity for yeast Sil1 has potential implications for the human ataxia Marinesco-Sjögren syndrome, where it is interesting to speculate that a disruption in ER redox-signaling (due to genetic defects in SIL1) may influence disease pathology.DOI: http://dx.doi.org/10.7554/eLife.24141.001

Role of the activation gate in determining the extracellular potassium dependency of block of HERG by trapped drugs, Channels, 7:1, 23-33,

KCNQ2 but not KNCQ3, is expressed on neuronal axons, where it might regulate action potential propagation or neurotransmitter release. Previously, we showed that Syntaxin 1A physically interacts with homomeric KCNQ2 in brain synaptosomes and in Xenopus oocytes. In oocytes, this interaction results in a reduction of the current's amplitude and reduction of the channel's activation rate. In vitro pull down revealed that Syntaxin 1A specifically binds the Helix A domain located in the C-terminus of both KCNQ2 and KCNQ3. However, binding of Syntaxin to Helix A does not mediate Syntaxin's effect on the channel. We propose that the N-trminus of KCNQ2 plays a major role in the syntaxin's modulation since substitution of the proximal N-terminus of KCNQ2 with that of KCNQ3 abolished this effect. Since the effect of syntaxin 1A is mediated through the N-terminus we assume that the N-C termini of the channel interact. To study this hypothesis we used florescence resonance energy transfer (FRET) experiments, single channel analysis and biochemical approaches using chimeric channels. Together, our results point toward an allosteric modulation of KCNQ2 gating by Syntaxin 1A, facilitated by N-C termini interaction. 2316-Pos Board B302Slowing of Instantaneous Inactivation Causes Macroscopic Current Decay in Kv7.1 Alanine Mutants Vitya Vardanyan, Lijuan Ma, Olaf Pongs. Alanine scanning mutagenesis in Kv7.1 S4-S5 linker and the pore region revealed mutant channels with fast current decay at permanent depolarizing pulses above 10mV. It has been previously shown that such a macroscopic inactivation of many Kv7.1 pore mutants occurs in parallel to instantaneous inactivation of Kv7.1 channel. We have chosen four mutant channels I268A, G269A, F339A and F340A with most pronounced current decays to investigate the molecular mechanism underling decay process. Inactivation of these channels could not be explained by intracellular Naþ block reported earlier for Kv7.1 wild type channel. The fast current decay kinetics significantly changed neither by varying the extracellular Kþ concentration nor by replacement of the Kþ with equimolar Rbþ. Recovery from inactivation showed fast and slow components. The slow component was accelerated at high extracellular Kþ conditions, whereas the fast component did not change significantly. Our data suggest that instantaneous inactivation of wild type Kv7.1 channel markedly slowed by above mentioned alanine mutations resulting in macroscopic decay of current during prolonged depolarization.

Protein folding in the endoplasmic reticulum (ER) has emerged recently as a source of reactive oxygen species (ROS). Despite a current appreciation of the potential for ER‐derived ROS to contribute to cell dysfunction, scientists lack a fundamental understanding of how the ER manages ROS production. Taking advantage of our ability to selectively create a hyper‐oxidized ER, we have identified a novel redox pathway that plays a central role in sensing and responding to redox imbalances within the ER. Key to this pathway is the lumenal chaperone BiP, a member of the Hsp70 ATPase family. Intriguingly, this BiP‐centric cellular response to ER oxidative stress is mechanistically distinct from previously characterized ER stress responses and involves redox signaling via a conserved BiP cysteine. The pronounced sensitivity of a cyteine‐less BiP mutant strain to oxidative stress suggests that alteration of the BiP cysteine (e.g. its oxidation) is part of a fundamental mechanism for cell protection. We suggest that post‐translational modification of the BiP cysteine alters BiP's catalytic cycle. We propose that alteration of BiP activity during oxidative stress allows the cell to cope with an existing misoxidized substrate load.