Kv2.1 exhibits two distinct forms of localization patterns on the neuronal plasma membrane: One population is freely diffusive and regulates electrical activity via voltage-dependent K conductance while a second one localizes to micrometer-sized clusters that contain densely packed, but nonconducting, channels. We have previously established that these clusters represent endoplasmic reticulum/plasma membrane (ER/PM) junctions that function as membrane trafficking hubs and that Kv2.1 plays a structural role in forming these membrane contact sites in both primary neuronal cultures and transfected HEK cells. Clustering and the formation of ER/PM contacts are regulated by phosphorylation within the channel C terminus, offering cells fast, dynamic control over the physical relationship between the cortical ER and PM. The present study addresses the mechanisms by which Kv2.1 and the related Kv2.2 channel interact with the ER membrane. Using proximity-based biotinylation techniques in transfected HEK cells we identified ER VAMP-associated proteins (VAPs) as potential Kv2.1 interactors. Confirmation that Kv2.1 and -2.2 bind VAPA and VAPB employed colocalization/redistribution, siRNA knockdown, and Förster resonance energy transfer (FRET)-based assays. CD4 chimeras containing sequence from the Kv2.1 C terminus were used to identify a noncanonical VAP-binding motif. VAPs were first identified as proteins required for neurotransmitter release in and are now known to be abundant scaffolding proteins involved in membrane contact site formation throughout the ER. The VAP interactome includes AKAPs, kinases, membrane trafficking machinery, and proteins regulating nonvesicular lipid transport from the ER to the PM. Therefore, the Kv2-induced VAP concentration at ER/PM contact sites is predicted to have wide-ranging effects on neuronal cell biology.

We established the importance of phosphorylation of cAMP responsive element-binding protein (CREB) to both the familiarity discrimination component of long-term recognition memory and plasticity within the perirhinal cortex of the temporal lobe. Adenoviral transduction of perirhinal cortex (and adjacent visual association cortex) with a dominant-negative inhibitor of CREB impaired the preferential exploration of novel over familiar objects at a long (24 h) but not a short (15 min) delay, disrupted the normal reduced activation of perirhinal neurons to familiar compared with novel pictures, and impaired long-term potentiation of synaptic transmission in perirhinal slices. The consistency of these effects across the behavioral, systems, and cellular levels of analysis provides strong evidence for involvement of CREB phosphorylation in synaptic plastic processes within perirhinal cortex necessary for long-term recognition memory.

Attachment of multiple chelated Gd3+ ions to the interior of bacteriophage P22 viral capsids afford nanoscale MRI contrast agents with extremely high relaxivity values. Highly fenestrated ‘wiffleball’ morphology is unique to P22 and assures water exchange between the environment and interior cavity of the capsid. The cavity of P22 ‘wiffleball’ was functionalized with a branched oligomer comprising of multiple DTPA-Gd complexes resulting in an impressive payload of 1,900 Gd3+ ions inside each 64nm capsid. High relaxivities of r1 ionic = 21.7 mM−1 sec−1 and r1 particle = 41,300 mM−1 sec−1 at 298K, 0.65T (28MHz) are reported, with r1/r2 ratio of 0.80 and optimized rotational correlation time for this system. Specific design modifications are suggested for future improvements of viral capsid-based MRI contrast agents directed toward clinical translation.

Voltage-gated sodium (Na v ) channels are responsible for the depolarizing phase of the action potential in most nerve cells, and Na v channel localization to the axon initial segment is vital to action potential initiation. Na v channels in the soma play a role in the transfer of axonal output information to the rest of the neuron and in synaptic plasticity, although little is known about Na v channel localization and dynamics within this neuronal compartment. This study uses single-particle tracking and photoactivation localization microscopy to analyze cell-surface Na v 1.6 within the soma of cultured hippocampal neurons. Mean-square displacement analysis of individual trajectories indicated that half of the somatic Na v 1.6 channels localized to stable nanoclusters~230 nm in diameter. Strikingly, these domains were stabilized at specific sites on the cell membrane for >30 min, notably via an ankyrin-independent mechanism, indicating that the means by which Na v 1.6 nanoclusters are maintained in the soma is biologically different from axonal localization. Nonclustered Na v 1.6 channels showed anomalous diffusion, as determined by mean-square-displacement analysis. High-density single-particle tracking of Na v channels labeled with photoactivatable fluorophores in combination with Bayesian inference analysis was employed to characterize the surface nanoclusters. A subpopulation of mobile Na v 1.6 was observed to be transiently trapped in the nanoclusters. Somatic Na v 1.6 nanoclusters represent a new, to our knowledge, type of Na v channel localization, and are hypothesized to be sites of localized channel regulation.

The potassium channels Kv2.1 and Kv2.2 are widely expressed throughout the mammalian brain. Kv2.1 provides the majority of delayed rectifying current in rat hippocampus while both channels are differentially expressed in cortex. Particularly unusual is their neuronal surface localization pattern: while half the channel population is freely-diffusive on the plasma membrane as expected from the generalized Singer & Nicolson fluid mosaic model, the other half localizes into micron-sized clusters on the soma, dendrites, and axon initial segment. These clusters contain hundreds of channels, which for Kv2.1, are largely non-conducting. Competing theories of the mechanism underlying Kv2.1 clustering have included static tethering to being corralled by an actin fence. Now, recent work has demonstrated channel clustering is due to formation of endoplasmic reticulum/plasma membrane (ER/PM) junctions through interaction with ER-resident VAMP-associated proteins (VAPs). Interaction between surface Kv2 channels and ER VAPs groups channels together in clusters. ER/PM junctions play important roles in inter-organelle communication: they regulate ion flux, are involved in lipid transfer, and are sites of endo- and exocytosis. Kv2-induced ER/PM junctions are regulated through phosphorylation of the channel C-terminus which in turn regulates VAP binding, providing a rapid means to create or dismantle these microdomains. In addition, insults such as hypoxia or ischemia disrupt this interaction resulting in ER/PM junction disassembly. Kv2 channels are the only known plasma membrane protein to form regulated, injury sensitive junctions in this manner. Furthermore, it is likely that concentrated VAPs at these microdomains sequester additional interactors whose functions are not yet fully understood.

Advances in three-dimensional secondary ion mass spectrometry (SIMS) imaging have enabled visualizing the subcellular distributions of various lipid species within individual cells. However, the difficulty of locating organelles using SIMS limits efforts to study their lipid compositions. Here, the authors have assessed whether endoplasmic reticulum (ER)-Tracker Blue White DPX, which is a commercially available stain for visualizing the endoplasmic reticulum using fluorescence microscopy, produces distinctive ions that can be used to locate the endoplasmic reticulum using SIMS. Time-of-flight-SIMS tandem mass spectrometry (MS) imaging was used to identify positively and negatively charged ions produced by the ER-Tracker stain. Then, these ions were used to localize the stain and thus the endoplasmic reticulum, within individual human embryonic kidney cells that contained higher numbers of endoplasmic reticulum-plasma membrane junctions on their surfaces. By performing MS imaging of selected ions in parallel with the precursor ion (MS) imaging, the authors detected a chemical interference native to the cell at the same nominal mass as the pentafluorophenyl fragment from the ER-Tracker stain. Nonetheless, the fluorine secondary ions produced by the ER-Tracker stain provided a distinctive signal that enabled locating the endoplasmic reticulum using SIMS. This simple strategy for visualizing the endoplasmic reticulum in individual cells using SIMS could be combined with existing SIMS methodologies for imaging intracellular lipid distribution and to study the lipid composition within the endoplasmic reticulum.

The endoplasmic reticulum (ER) forms a continuous and dynamic network throughout a neuron, extending from dendrites to axon terminals, and axonal ER dysfunction is implicated in several neurological disorders. In addition, tight junctions between the ER and plasma membrane (PM) are formed by several molecules including Kv2 channels, but the cellular functions of many ER-PM junctions remain unknown. Recently, dynamic Ca 2+ uptake into the ER during electrical activity was shown to play an essential role in synaptic transmission. Our experiments demonstrate that Kv2.1 channels are necessary for enabling ER Ca 2+ uptake during electrical activity, as knockdown (KD) of Kv2.1 rendered both the somatic and axonal ER unable to accumulate Ca 2+ during electrical stimulation. Moreover, our experiments demonstrate that the loss of Kv2.1 in the axon impairs synaptic vesicle fusion during stimulation via a mechanism unrelated to voltage. Thus, our data demonstrate that a nonconducting role of Kv2.1 exists through its binding to the ER protein VAMP-associated protein (VAP), which couples ER Ca 2+ uptake with electrical activity. Our results further suggest that Kv2.1 has a critical function in neuronal cell biology for Ca 2+ handling independent of voltage and reveals a critical pathway for maintaining ER lumen Ca 2+ levels and efficient neurotransmitter release. Taken together, these findings reveal an essential nonclassical role for both Kv2.1 and the ER-PM junctions in synaptic transmission.

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