Probing Subunits Interactions in K Channels Using Photo-Crosslinking via Genetically Encoded p-Azido-L-phenylalanine.

Potassium channels are multimeric protein complexes regulated by diverse physiological and pharmacological ligands. The key to understanding mechanisms of channel regulation is the ability to detect structural changes associated with ligand binding. While high-resolution structural methods such as X-ray crystallography and single-particle cryo-electron microscopy offer direct visualization of channel structures, these methods do have limitations and may not be suitable for the question of interest.

Pharmacological Correction of Trafficking Defects in ATP-sensitive Potassium Channels Caused by Sulfonylurea Receptor 1 Mutations.

ATP-sensitive potassium (K) channels play a key role in mediating glucose-stimulated insulin secretion by coupling metabolic signals to β-cell membrane potential. Loss of K 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.

The Ribosome-Sec61 Translocon Complex Forms a Cytosolically Restricted Environment for Early Polytopic Membrane Protein Folding.

Transmembrane topology of polytopic membrane proteins (PMPs) is established in the endoplasmic reticulum (ER) by the ribosome Sec61-translocon complex (RTC) through iterative cycles of translocation initiation and termination. It remains unknown, however, whether tertiary folding of transmembrane domains begins after the nascent polypeptide integrates into the lipid bilayer or within a proteinaceous environment proximal to translocon components. To address this question, we used cysteine scanning mutagenesis to monitor aqueous accessibility of stalled translation intermediates to determine when, during biogenesis, hydrophilic peptide loops of the aquaporin-4 (AQP4) water channel are delivered to cytosolic and lumenal compartments.

Cotranslational stabilization of Sec62/63 within the ER Sec61 translocon is controlled by distinct substrate-driven translocation events.

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.

Structurally distinct ligands rescue biogenesis defects of the KATP channel complex via a converging mechanism.

Small molecules that correct protein misfolding and misprocessing defects offer a potential therapy for numerous human diseases. However, mechanisms underlying pharmacological correction of such defects, especially in heteromeric complexes with structurally diverse constituent proteins, are not well understood. Here we investigate how two chemically distinct compounds, glibenclamide and carbamazepine, correct biogenesis defects in ATP-sensitive potassium (KATP) channels composed of sulfonylurea receptor 1 (SUR1) and Kir6.

Carbamazepine inhibits ATP-sensitive potassium channel activity by disrupting channel response to MgADP.

In pancreatic β-cells, K(ATP) channels consisting of Kir6.2 and SUR1 couple cell metabolism to membrane excitability and regulate insulin secretion. Sulfonylureas, insulin secretagogues used to treat type II diabetes, inhibit K(ATP) channel activity primarily by abolishing the stimulatory effect of MgADP endowed by SUR1.

Pharmacological rescue of trafficking-impaired ATP-sensitive potassium channels.

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.

Semisynthetic K+ channels show that the constricted conformation of the selectivity filter is not the C-type inactivated state.

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.

Polyol osmolytes stabilize native-like cooperative intermediate state of yeast hexokinase A at low pH.

Osmolytes produced under stress in animal and plant systems have been shown to increase thermal stability of the native state of a number of proteins as well as induce the formation of molten globule (MG) in acid denatured states and compact conformations in natively unfolded proteins. However, it is not clear whether these solutes stabilize native state relative to the MG state under partially denaturing conditions. Yeast hexokinase A exists as a MG state at pH 2.

In vitro folding of KvAP, a voltage-gated K+ channel.

In this contribution, we report in vitro folding of the archaebacterial voltage-gated K(+) channel, K(v)AP. We show that in vitro folding of the K(v)AP 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.

Stepwise insertion and inversion of a type II signal anchor sequence in the ribosome-Sec61 translocon complex.

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: (1) head-first insertion into Sec61α, (2) nascent chain accumulation within the RTC, (3) inversion from type I to type II topology, and (4) 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.