COMMENTARY Kv2 potassium channels meet VAP COMMENTARY Elizabeth Wen Suna,b,c,d and Pietro De Camillia,b,c,d,1 A defining characteristic of eukaryotic cells is the presence of distinct intracellular membrane-bound A compartments. Much research has focused on the functional interconnection of these organelles via membrane traffic. A flurry of recent studies, however, has brought to center stage the important role of interorganelle communication independent of vesic- ular transport and mediated by direct contacts (1–3). At these sites, membranes are tethered to each other by protein–protein or protein–lipid interactions not leading to fusion. These contacts play a variety of func- tions, including regulation of ion fluxes across mem- Plasma Membrane Endoplasmic Reticulum ER-PM contact sites (<30nm) branes and transport of lipids between participating organelles. In PNAS, Johnson et al. (4) provide yet an- cytosolic VAP interactors other unexpected example of direct communication B Kv2 channels B’ VAP (A/B) PM B’ between two membranes: the binding of the plasma Mitochondrion Kv2 membrane (PM)-localized major delayed-rectifier voltage- PM + gated K channels, Kv2.1 and Kv2.2, to VAMP-associated Endosome C’ protein (VAP), an integral membrane protein of the endo- Lipid droplet N’ plasmic reticulum (ER). VAP Kv2.1 and Kv2.2 (KCNB1 and KCNB2) channels RyR are very abundant in the brain, where they play a major MVB role in neuronal excitability. They form large clusters in Golgi PRC region ER the PM of neuronal cell bodies, proximal dendrites, and ER Lipid transfer axon initial segments, and such clusters are at sites – where the PM is tightly apposed to the ER (5). In the Fig. 1. ER PM contacts and role of VAP as a tethering protein at the surface of the ER. (A) A 3D reconstruction from a focused ion beam–SEM image stack of the ER in clusters, the cytosolic tails of the channels are in a phos- the cortical region of the cell body of a mouse brain neuron. ER cisternae closely phorylated state and the clusters disperse within mi- apposed to the PM are shown in red (adapted with permission from ref. 8). (Scale nutes due to calcium-dependent dephosphorylation bar: 400 nm.) (B) Schematic cartoon highlighting VAP-dependent interactions at – upon excessive excitatory stimulation or exposure to the ER surface and the coclustering of VAP with Kv2 channels at ER PM contacts. (B′) The cartoon highlights binding of a VAP dimer to the PRC region in the noxious conditions, such as hypoxia (6). While an amino C-terminal portion of Kv2 channels and to a lipid transport protein. A RyR is acid stretch, referred to as the PRC (proximal restriction also shown. Only one subunit of the tetrameric Kv2 channels and two subunits and clustering), was known to be required for clustering of the RyR are shown for simplicity. (7), how the PRC mediates clustering remained unclear. The ER is the most abundant intracellular mem- branous organelle. It comprises a system of tubules cellular membranes (Fig. 1) (8). Contacts between the and cisterns that are continuous with each other and ER and the PM were first observed in muscle, where populate every cell compartment, including axon they are the basis for depolarization contraction cou- endings and dendritic spines in neurons (8). Its mem- pling via a direct interaction between PM voltage- + branes make focal and tight apposition with all other dependent Ca2 channels and the ryanodine receptors aDepartment of Neuroscience, Yale University School of Medicine, New Haven, CT 06510; bDepartment of Cell Biology, Yale University School of Medicine, New Haven, CT 06510; cKavli Institute for Neuroscience, Yale University School of Medicine, New Haven, CT 06510; and dHoward Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510 Author contributions: E.W.S. and P.D.C. wrote the paper. The authors declare no conflict of interest. Published under the PNAS license. See companion article on page E7331. 1To whom correspondence should be addressed. Email: [email protected]. Published online July 17, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1810059115 PNAS | July 31, 2018 | vol. 115 | no. 31 | 7849–7851 Downloaded by guest on October 2, 2021 + (RyR): that is, the Ca2 release channels of the ER. However, it more of its serines, providing a possible explanation for why subsequently became clear that ER–PM contacts are present in the clustering of Kv2 channels is critically dependent on their all cells and may have a variety of functions. For example, ER–PM being in the phosphorylated state. Supporting this possibility, + contacts mediate store-operated Ca2 entry, a housekeeping pro- serines within the PRC (S583, S586, and S589) had been previ- + cess in which binding of the ER protein STIM1 (a sensor of Ca2 ously shown to be required for clustering (7), although a precise + concentration in the ER lumen) to the PM Ca2 channel Orai trig- validation of this mode of binding will require structural studies. A + gers Ca2 influx. Another function is lipid transport, or countertran- puzzling question left open by these studies is what controls the sport, via proteins that contain lipid transport modules and also clustering of Kv2 channels at axon initial segments, because the tether the two participating membranes (1). This transport helps PRC region of Kv2, and thus the interaction with VAP, is dispens- control the lipid composition of the PM and allows homeostatic able for this localization (13). adaptations of its lipid bilayer in response to acute perturbations. While the study by Johnson et al. (4) provides an explanation ER–PM appositions are particularly large in neurons, where for the clustering of Kv2 channels at ER–PM contacts in neuronal micrometer-sized ER cisternae (subsurface/hypolemmal cisternae), often with a very narrow lumen (thin ER), cover about 10% of the PM of cell bodies (Fig. 1A) (8). Smaller contacts are also present in Collectively, Johnson et al.’s experiments dendrites, axons, and axon terminals. demonstrate the causality of Kv2 channels The localization of Kv2 clusters at ER–PM contacts suggested their direct or indirect interactions with ER proteins. To identify binding to VAP for their accumulation, and thus such partners, Johnson et al. (4) capitalize on proximity labeling clustering, at ER–PM contact sites. with biotin using APEX-based methodology (8). They appended APEX to the cytosolic region of AMIGO, an integral plasma mem- brane protein closely associated with the α-subunits of the Kv2 chan- perikarya and dendrites, the physiological significance of this nels, and then searched for biotinylated proteins by streptavidin localization, and more specifically of the interaction with VAP, labeling. A prominently biotinylated protein of 33 kDa was identified, remains an open question. Clustered Kv2 channels have a leading Johnson et al. (4) to hypothesize that VAP (also known as different threshold for activation than nonclustered channels VAP33), an abundant ER protein, could be the interactor (9). Johnson (14), raising the possibility that binding to VAP may control et al. (4) tested and proved this hypothesis through expression and their voltage sensitivity. Alternatively, as VAP proteins dimerize coexpression of tagged wild-type and mutant Kv2 channels and and further oligomerize (10), they could recruit other proteins VAP proteins, as well as through knockdown experiments in neu- to help modulate channel function indirectly. They may do so rons and HEK293 cells. Collectively, Johnson et al.’s experiments by affecting the focal lipid composition of the PM, as a large demonstrate the causality of Kv2 channels binding to VAP for their fraction of client proteins for VAP are implicated in lipid accumulation, and thus clustering, at ER–PM contact sites (Fig. 1B′). transport and metabolism and the function of Kv2 channels is – VAP is the collective name for two very similar small dimeric regulated by PI(4,5)P2 (9, 15). For example, Kv2.1-enriched ER proteins expressed throughout evolution from yeast (Scs2 and PM contacts that face C-type presynaptic nerve terminals are Scs22) to humans (VAPA and VAPB), and comprising an N-terminal also enriched in phospholipase C (PLC)-coupled muscarinic major sperm protein (MSP) module followed by a coiled–coil region acetylcholine receptors (16), thus making plausible a potential and a C-terminal hydrophobic sequence that anchors it to the ER. coupling between PI(4,5)P2 metabolism and Kv2.1 channel The surface of the MSP domain harbors a positively charged function. Clearly, many other scenarios are possible, as a groove that functions as the binding site for the so-called FFAT recent comprehensive analysis of the VAP “interactome” motif [two phenylalanine (F) in an acidic tract] (9, 10). Dozens of revealed many proteins with roles not obviously linked to lipid proteins with this motif or its variants have been identified and for dynamics (9). many of them the interaction with VAP has been experimentally VAP-induced recruitment of the ER to the Kv2 channels in the confirmed (9). Thus, VAP serves as an anchor at the surface of the PM may also have roles independent of VAP itself. In Purkinje + ER for many cytosolic proteins, and participates in bridging two cells, another class of K channels, the BK channels (large- + membranes when the FFAT motif-containing partner protein is in conductance Ca2 -activated potassium channels), is concen- turn anchored via a lipid-binding module or a transmembrane re- trated in regions of the PM closely apposed to the ER (17). + gion to another membrane (Fig. 1 B and B′). The importance of VAP The association of K channels with the ER may provide a func- + for cell physiology is underscored by the striking perturbation of tional link between these channels and Ca2 dynamics.