The endoplasmic reticulum (ER) and plasma membrane (PM) form junctions crucial to ion and lipid signaling and homeostasis. The Kv2.1 ion channel is localized at ER–PM junctions in brain neurons and is unique among PM proteins in its ability to remodel these specialized membrane contact sites. Here, we show that this function is conserved between Kv2.1 and Kv2.2, which differ in their biophysical properties, modulation, and cellular expression. Kv2.2 ER–PM junctions are present at sites deficient in the actin cytoskeleton, and disruption of the actin cytoskeleton affects their spatial organization. Kv2.2-containing ER–PM junctions overlap with those formed by canonical ER–PM tethers. The ability of Kv2 channels to remodel ER–PM junctions is unchanged by point mutations that eliminate their ion conduction but eliminated by point mutations within the Kv2-specific proximal restriction and clustering (PRC) domain that do not impact their ion channel function. The highly conserved PRC domain is sufficient to transfer the ER–PM junction–remodeling function to another PM protein. Last, brain neurons in Kv2 double-knockout mice have altered ER–PM junctions. Together, these findings demonstrate a conserved in vivo function for Kv2 family members in remodeling neuronal ER–PM junctions that is distinct from their canonical role as ion-conducting channels shaping neuronal excitability.

Abstract:The use of chemical ligation within the realm of peptide chemistry has opened various opportunities to expand the applications of peptides/proteins in biological sciences. Expansion and refinement of ligation chemistry has made it possible for the entry of peptides into the world of viable oral therapeutic drugs through peptide backbone cyclization. This progression has been a journey of chemical exploration and transition, leading to the dominance of native chemical ligation in the present advances of peptide/protein applications. Here we illustrate and explore the historical and current nature of peptide ligation, providing a clear indication to the possibilities and use of these novel methods to take peptides outside their typically defined boundaries.

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Ion channels are polymorphic membrane proteins whose high-resolution structures offer images of individual conformations, giving us starting points for identifying the complex and transient allosteric changes that give rise to channel physiology. Here, we report live-cell imaging of voltage-dependent structural changes of voltage-gated Kv2.1 channels using peptidyl tarantula toxins labeled with an environment-sensitive fluorophore, whose spectral shifts enable identification of voltage-dependent conformation changes in the resting voltage sensing domain (VSD) of the channel. We synthesize a new environment-sensitive, far-red fluorophore, julolidine phenoxazone (JP) azide, and conjugate it to tarantula toxin GxTX to characterize Kv2.1 VSD allostery during membrane depolarization. JP has an inherent response to the polarity of its immediate surroundings, offering site-specific structural insight into each channel conformation. Using voltage-clamp spectroscopy to collect emission spectra as a function of membrane potential, we find that they vary with toxin labeling site, the presence of Kv2 channels, and changes in membrane potential. With a high-affinity conjugate in which the fluorophore itself interacts closely with the channel, the emission shift midpoint is 50 mV more negative than the Kv2.1 gating current midpoint. This suggests that substantial conformational changes at the toxin–channel interface are associated with early gating charge transitions and these are not concerted with VSD motions at more depolarized potentials. These fluorescent probes enable study of conformational changes that can be correlated with electrophysiology, putting channel structures and models into a context of live-cell membranes and physiological states.