Summary In eukaryotic cells, the ribosome-Sec61 translocon complex (RTC) establishes membrane protein topology by cotranslationally partitioning nascent polypeptides into the cytosol, ER lumen, and lipid bilayer. Using photocrosslinking, collisional quenching, cysteine accessibility and protease protection, we show that a canonical type II signal anchor (SA) acquires its topology through four tightly coupled and mechanistically distinct steps: i) head-first insertion into Sec61α, ii) nascent chain accumulation within the RTC, iii) inversion from type I to a type II topology, and iv) stable translocation of C-terminal flanking residues. Progression through each stage is induced by incremental increases in chain length and involves abrupt changes in the molecular environment of the SA. Importantly, type II SA inversion deviates from a type I SA at an unstable intermediate whose topology is controlled by dynamic interactions between the ribosome and translocon. Thus, the RTC coordinates SA topogenesis within a protected environment via sequential energetic transitions of the TM segment.

SUMMARY The ER Sec61 translocon is a large macromolecular machine responsible for partitioning secretory and membrane polypeptides into the lumen, cytosol, and lipid bilayer. Because the Sec61 protein-conducting channel has been isolated in multiple membrane-derived complexes, we determined how the nascent polypeptide modulates translocon component associations during defined cotranslational translocation events. The model substrate preprolactin (pPL) was isolated principally with Sec61αβγ upon membrane targeting, whereas higher-order complexes containing OST, TRAP, and TRAM were stabilized following substrate translocation. Blocking pPL translocation by passenger domain folding favored stabilization of an alternate complex that contained Sec61, Sec62 and Sec63. Moreover, Sec62/63 stabilization within the translocon occurred for native endogenous substrates, such as the prion protein, and correlated with a delay in translocation initiation. This data shows that cotranslational translocon contacts are ultimately controlled by the engaged nascent chain and the resultant substrate-driven translocation events.

Background: Carbamazepine and glibenclamide correct K ATP channel trafficking defects. Results: Carbamazepine and glibenclamide share a binding pocket in the channel and enhance cross-linking of Kir6.2 to SUR1. Conclusion: The two structurally distinct drugs correct K ATP channel biogenesis defects caused by mutations in SUR1 and Kir6.2 by promoting interactions between the two channel subunits. Significance: The heteromeric subunit interface is an important target for pharmacological chaperones.

C-type inactivation of K + channels plays a key role in modulating cellular excitability. During C-type inactivation, the selectivity filter of a K + channel changes conformation from a conductive to a nonconductive state. Crystal structures of the KcsA channel determined at low K + or in the open state revealed a constricted conformation of the selectivity filter, which was proposed to represent the C-type inactivated state. However, structural studies on other K + channels do not support the constricted conformation as the C-type inactivated state. In this study, we address whether the constricted conformation of the selectivity filter is in fact the C-type inactivated state. The constricted conformation can be blocked by substituting the first conserved glycine in the selectivity filter with the unnatural amino acid D-Alanine. Protein semisynthesis was used to introduce D-Alanine into the selectivity filters of the KcsA channel and the voltage-gated K + channel K v AP. For semisynthesis of the K v AP channel, we developed a modular approach in which chemical synthesis is limited to the selectivity filter whereas the rest of the protein is obtained by recombinant means. Using the semisynthetic KcsA and K v AP channels, we show that blocking the constricted conformation of the selectivity filter does not prevent inactivation, which suggests that the constricted conformation is not the C-type inactivated state.

ATP-sensitive potassium (K ATP ) channels play a key role in mediating glucose-stimulated insulin secretion by coupling metabolic signals to ␤-cell membrane potential. Loss of K ATP channel function due to mutations in ABCC8 or KCNJ11, genes encoding the sulfonylurea receptor 1 (SUR1) or the inwardly rectifying potassium channel Kir6.2, respectively, results in congenital hyperinsulinism. Many SUR1 mutations prevent trafficking of channel proteins from the endoplasmic reticulum to the cell surface. Channel inhibitors, including sulfonylureas and carbamazepine, have been shown to correct channel trafficking defects. In the present study, we identified 13 novel SUR1 mutations that cause channel trafficking defects, the majority of which are amenable to pharmacological rescue by glibenclamide and carbamazepine. By contrast, none of the mutant channels were rescued by K ATP channel openers. Cross-linking experiments showed that K ATP channel inhibitors promoted interactions between the N terminus of Kir6.2 and SUR1, whereas channel openers did not, suggesting the inhibitors enhance intersubunit interactions to overcome channel biogenesis and trafficking defects. Functional studies of rescued mutant channels indicate that most mutants rescued to the cell surface exhibited WT-like sensitivity to ATP, MgADP, and diazoxide. In intact cells, recovery of channel function upon trafficking rescue by reversible sulfonylureas or carbamazepine was facilitated by the K ATP channel opener diazoxide. Our study expands the list of K ATP channel trafficking mutations whose function can be recovered by pharmacological ligands and provides further insight into the structural mechanism by which channel inhibitors correct channel biogenesis and trafficking defects.Protein function relies on the proper folding, assembly, and trafficking to specific cellular compartments. In the case of plasma membrane proteins, such as ion channels and receptors, they must pass quality surveillance in the endoplasmic reticulum (ER) 3 to enter the secretory pathway and ultimately reach the cell surface. Numerous diseases arise due to mutations that disrupt protein folding, assembly, and subsequent trafficking to the cell surface (1), hereafter referred to as trafficking mutations. Small molecules termed pharmacological chaperones hold promise as a means of therapy for such diseases by interacting with mutant proteins and correcting their folding and trafficking defects (2-4).Congenital hyperinsulinism (HI) is a rare, life-threatening disease characterized by persistent insulin secretion despite extreme hypoglycemia (5). The most common cause of HI is loss-of-function mutations in the ABCC8 or KCNJ11 genes encoding the sulfonylurea receptor 1 (SUR1) and inwardly rectifying potassium channel Kir6.2 proteins, respectively (5-7). SUR1 and Kir6.2 form the pancreatic subtype of the ATP-sensitive K ϩ (K ATP ) channel, which plays a key role in glucosestimulated insulin secretion by coupling glucose metabolism to ␤-cell membrane excitability (7-9). SUR1 or Kir...

ATP-sensitive potassium (KATP) channels link cell metabolism to membrane excitability and are involved in a wide range of physiological processes including hormone secretion, control of vascular tone, and protection of cardiac and neuronal cells against ischemic injuries. In pancreatic β-cells, KATP channels play a key role in glucose-stimulated insulin secretion, and gain or loss of channel function results in neonatal diabetes or congenital hyperinsulinism, respectively. The β-cell KATP channel is formed by co-assembly of four Kir6.2 inwardly rectifying potassium channel subunits encoded by KCNJ11 and four sulfonylurea receptor 1 subunits encoded by ABCC8. Many mutations in ABCC8 or KCNJ11 cause loss of channel function, thus, congenital hyperinsulinism by hampering channel biogenesis and hence trafficking to the cell surface. The trafficking defects caused by a subset of these mutations can be corrected by sulfonylureas, KATP channel antagonists that have long been used to treat type 2 diabetes. More recently, carbamazepine, an anticonvulsant that is thought to target primarily voltage-gated sodium channels has been shown to correct KATP channel trafficking defects. This article reviews studies to date aimed at understanding the mechanisms by which mutations impair channel biogenesis and trafficking and the mechanisms by which pharmacological ligands overcome channel trafficking defects. Insight into channel structure-function relationships and therapeutic implications from these studies are discussed.

In this contribution, we report in vitro folding of the archaebacterial voltage gated K+ channel, KvAP. We show that in vitro folding of the KvAP channel from the extensively unfolded state requires lipid vesicles and that the refolded channel is biochemically and functionally similar to the native channel. The in vitro folding process is slow at room temperature and the folding yield depends on the composition of the lipid bilayer. The major factor influencing refolding is temperature and almost quantitative refolding of the KvAP channel is observed at 80 °C. In order to differentiate between insertion into the bilayer and folding within the bilayer, we developed a cysteine protection assay. Using this assay, we demonstrate that insertion of the unfolded protein into the bilayer is relatively fast at room temperature and independent of lipid composition suggesting that temperature and bilayer composition influence folding within the bilayer. Further, we demonstrate that in vitro folding provides an effective method for obtaining high yields of the native channel. Our studies suggest that the KvAP channel provides a good model system to investigate the folding of a multi-domain integral membrane protein.

Membrane proteins are structurally characterized in membrane mimetic environments, environments that may or may not reflect the properties of native membrane proteins. This is critically important since the structure of membrane proteins is influenced by the protein's environment. Here we will show structural data from magic angle spinning solid state NMR of the full length M2 protein from Influenza A observed in E. coli membranes. This protein has never been exposed to the denaturing influence of detergents nor to the length isolation, purification and reconstitution protocols that typify membrane protein sample preparation for structural studies. The data will be compared to the purified full length protein in synthetic liposomes and to spectra of smaller constructs for which there is a high resolution structure in lipid bilayers.