Mouse anti-Human KCNE1 monoclonal antibody, clone 6C23

The slow delayed rectifier potassium current (I-Ks) is a key repolarizing current during the cardiac action potential. It consists of four KCNQ1 alpha-subunits and up to four KCNE1 beta-subunits, which are thought to reside within external clefts of the channel. The interaction of KCNE1 with KCNQ1 dramatically delays opening of the channel but the mechanisms by which this occur are not yet fully understood. Here, we have used unnatural amino acid photo-cross-linking to investigate the dynamic interactions that occur between KCNQ1 and KCNE1 during activation gating. The unnatural amino acid p-Benzoylphenylalanine was successfully incorporated into two residues within the transmembrane domain of KCNE1: F56 and F57. UV-induced cross-linking suggested that F56Bpa interacts with KCNQ1 in the open state, whereas F57Bpa interacts predominantly in resting channel conformations. When UV was applied at progressively more depolarized preopen holding potentials, cross-linking of F57Bpa with KCNQ1 was slowed, which indicates that KCNE1 is displaced within the channel's cleft early during activation, or that conformational changes in KCNQ1 alter its interaction with KCNE1. In E1R/R4E KCNQ1, a mutant with constitutively activated voltage sensors, F56Bpa and F57Bpa KCNE1 were cross-linked in open and closed states, respectively, which suggests that their actions are mediated mainly by modulation of KCNQ1 pore function.

The elucidation of the structures and interactions of proteins and lipids in intact biological membranes remains largely uncharted territory. However, this information is crucial for understanding how organelles are assembled and how transmembrane machines transduce signals. The challenge of seeing how lipids and proteins engage each other in vivo remains difficult but is being aided by a group of amphipathic copolymers that spontaneously fragment native membranes into native nanodiscs. Poly(styrene-co-maleic acid) (SMA) copolymers have proven adept at converting membranes, cells and tissues directly into SMA lipid particles (SMALPs), allowing endogenous lipid: protein complexes to be prepared and analyzed. Unlike other amphipathic polymers such as amphipols, SMALP formation requires no conventional detergents, which typically strip lipid molecules from proteins and can destabilize multimers. A collaborative community of hundreds of investigators known as the SMALP network has emerged to develop and apply new technologies and identify new challenges and design potential solutions. In this contribution, we review recent practices and progress, focusing on novel SMA copolymers, modifications, alternatives and mechanisms. In addition, a brief overview will be provided, with reference to the further contributions to this special issue on the SMALP technology.