Elizabeth J. Akin

Kv2.1 cell surface clusters are insertion platforms for ion channel delivery to the plasma membrane

Kv2.1 surface clusters in transfected HEK cells and hippocampal neurons are shown to be trafficking platforms involved in potassium channel movement to and from the cell surface. This work is the first to define stable cell surface sites for ion channel delivery and retrieval at the cell surface.

Induction of stable ER-plasma-membrane junctions by Kv2.1 potassium channels.

ABSTRACT Junctions between cortical endoplasmic reticulum (cER) and the plasma membrane are a subtle but ubiquitous feature in mammalian cells; however, very little is known about the functions and molecular interactions that are associated with neuronal ER–plasma-membrane junctions. Here, we report that Kv2.1 (also known as KCNB1), the primary delayed-rectifier K+ channel in the mammalian brain, induces the formation of ER–plasma-membrane junctions. Kv2.1 localizes to dense, cell-surface clusters that contain non-conducting channels, indicating that they have a function that is unrelated to membrane-potential regulation. Accordingly, Kv2.1 clusters function as membrane-trafficking hubs, providing platforms for delivery and retrieval of multiple membrane proteins. Using both total internal reflection fluorescence and electron microscopy we demonstrate that the clustered Kv2.1 plays a direct structural role in the induction of stable ER–plasma-membrane junctions in both transfected HEK 293 cells and cultured hippocampal neurons. Glutamate exposure results in a loss of Kv2.1 clusters in neurons and subsequent retraction of the cER from the plasma membrane. We propose Kv2.1-induced ER–plasma-membrane junctions represent a new macromolecular plasma-membrane complex that is sensitive to excitotoxic insult and functions as a scaffolding site for both membrane trafficking and Ca2+ signaling.

Preferential Targeting of Nav1.6 Voltage-Gated Na+ Channels to the Axon Initial Segment during Development

During axonal maturation, voltage-gated sodium (Nav) channels accumulate at the axon initial segment (AIS) at high concentrations. This localization is necessary for the efficient initiation of action potentials. The mechanisms underlying channel trafficking to the AIS during axonal development have remained elusive due to a lack of Nav reagents suitable for high resolution imaging of channels located specifically on the cell surface. Using an optical pulse-chase approach in combination with a novel Nav1.6 construct containing an extracellular biotinylation domain we demonstrate that Nav1.6 channels are preferentially inserted into the AIS membrane during neuronal development via direct vesicular trafficking. Single-molecule tracking illustrates that axonal channels are immediately immobilized following delivery, while channels delivered to the soma are often mobile. Neither a Nav1.6 channel lacking the ankyrin-binding motif nor a chimeric Kv2.1 channel containing the Nav ankyrinG-binding domain show preferential AIS insertion. Together these data support a model where ankyrinG-binding is required for preferential Nav1.6 insertion into the AIS plasma membrane. In contrast, ankyrinG-binding alone does not confer the preferential delivery of proteins to the AIS.

Plasma membrane domains enriched in cortical endoplasmic reticulum function as membrane protein trafficking hubs

This study investigates the hypothesis that trafficking of membrane proteins occurs at plasma membrane (PM) domains adjacent to underlying cortical endoplasmic reticulum (cER). The authors observe exocytosis of transferrin receptor and vesicular stomatitis virus G-protein to occur preferentially (>80%) at cER-enriched PM domains. They also report a preferential (>80%) localization of clathrin-coated pits at these domains.

Ergodicity breaking on the neuronal surface emerges from random switching between diffusive states

Stochastic motion on the surface of living cells is critical to promote molecular encounters that are necessary for multiple cellular processes. Often the complexity of the cell membranes leads to anomalous diffusion, which under certain conditions it is accompanied by non-ergodic dynamics. Here, we unravel two manifestations of ergodicity breaking in the dynamics of membrane proteins in the somatic surface of hippocampal neurons. Three different tagged molecules are studied on the surface of the soma: the voltage-gated potassium and sodium channels Kv1.4 and Nav1.6 and the glycoprotein CD4. In these three molecules ergodicity breaking is unveiled by the confidence interval of the mean square displacement and by the dynamical functional estimator. Ergodicity breaking is found to take place due to transient confinement effects since the molecules alternate between free diffusion and confined motion.

Elucidating distinct ion channel populations on the surface of hippocampal neurons via single-particle tracking recurrence analysis

Protein and lipid nanodomains are prevalent on the surface of mammalian cells. In particular, it has been recently recognized that ion channels assemble into surface nanoclusters in the soma of cultured neurons. However, the interactions of these molecules with surface nanodomains display a considerable degree of heterogeneity. Here, we investigate this heterogeneity and develop statistical tools based on the recurrence of individual trajectories to identify subpopulations within ion channels in the neuronal surface. We specifically study the dynamics of the K^{+} channel Kv1.4 and the Na^{+} channel Nav1.6 on the surface of cultured hippocampal neurons at the single-molecule level. We find that both these molecules are expressed in two different forms with distinct kinetics with regards to surface interactions, emphasizing the complex proteomic landscape of the neuronal surface. Further, the tools presented in this work provide new methods for the analysis of membrane nanodomains, transient confinement, and identification of populations within single-particle trajectories.

Size of Cell-Surface Kv2.1 Domains is Governed by Growth Fluctuations

The Kv2.1 voltage-gated potassium channel forms stable clusters on the surface of different mammalian cells. Even though these cell-surface structures have been observed for almost a decade, little is known about the mechanism by which cells maintain them. We measure the distribution of domain sizes to study the kinetics of their growth. Using a Fokker-Planck formalism, we find no evidence for a feedback mechanism present to maintain specific domain radii. Instead, the size of Kv2.1 clusters is consistent with a model where domain size is established by fluctuations in the trafficking machinery. These results are further validated using likelihood and Akaike weights to select the best model for the kinetics of domain growth consistent with our experimental data.

Another piece to the intracellular FGF/Na+ channel puzzle

Neuronal communication requires the propagation of precisely regulated action potentials. Although a myriad of ion channels and modulatory proteins contribute to the action potential waveform and firing properties of each neuron, voltage-gated sodium (Nav) channels typically generate the crucial depolarizing event. Early Nav channel biochemistry identified two beta subunits (1), thus generating excitement over how accessory proteins might be involved in Nav channel physiology. In recent years, Nav channel interactors have grown to include intracellular fibroblast growth factor homologous factors (iFGFs) (for a recent review of this subject, see ref. 2). Although the majority of FGFs are secreted growth factors, a four-member subfamily now designated FGF11-14 is distinguished by generating nonsecreted proteins that do not interact with FGF receptors. Pioneering efforts by Waxman and colleagues (3, 4) demonstrated that these noncanonical FGFs directly bind the C terminus of Nav channels and influence both current density and gating properties. The current literature includes reports of numerous FGF12-14 interactions with various Nav alpha subunits. The picture is far from complete, however, as expression and functional effects vary not only depending on the FGF and Nav isoform partners but also on the FGF splice variant and cell background (2). Although this diversity has clearly complicated the field, FGFs’ role as Nav channel interacting proteins has been solidified by the discovery of a highly conserved Nav interaction site within the FGF core domain that interacts with specific amino acids within the Nav C termini (2). FGF14 and Nav channels are enriched at the axon initial segment (AIS) of several neuronal types, whereas FGF13 colocalizes with Nav1.6 at nodes of Ranvier of dorsal roots of primary afferents (5). Of note is that nodal localization was seen using a pan FGF13 antibody, … [↵][1]1To whom correspondence should be addressed. Email: michael.tamkun{at}colostate.edu. [1]: #xref-corresp-1-1

Single-Particle Tracking of Nav1.6 Demonstrates Different Mechanisms for Sodium Channel Anchoring within the AIS versus the Soma of Hippocampal Neurons

Voltage-gated sodium channels are responsible for the initiation of action potentials in excitable cells. These channels are highly concentrated at the axon initial segment (AIS) of neurons due to their interactions with ankyrin-G. This interaction is mediated by a 9 amino acid sequence, termed the Ankyrin Binding Motif (ABM) present on the II-III linker. In order to study the dynamics of sodium channels in living neurons in real time, we created a fluorescently labeled Nav1.6 protein with an extracellular tag (biotin acceptor domain). We used single-particle tracking of channels labeled with streptavidin conjugated quantum dots (QDs) and/or Alexa594 to directly compare the mobility of Nav1.6 channels localized to the AIS and somatodendritic compartments of 8DIV hippocampal neurons. We observed two populations of Nav1.6 channels, a small mobile population and a much larger immobile population. The mobile channels on the soma had a diffusion coefficient of 0.016 ± 0.008 μm2/s. To determine the role of ankyrin-G binding in the diffusion of the full-length sodium channel, we deleted the ABM from the Nav1.6 construct. As expected, this mutant channel did not concentrate at the AIS and instead was localized throughout the soma and processes, based on both GFP fluorescence and labeling of surface channels using streptavidin conjugated-Alexa594. Single-particle tracking of the mutant channels revealed that the majority of these channels (∼80%) are also immobile in the plasma membrane of the soma and dendrites. This suggests that although binding to ankyrin-G is necessary and sufficient for Nav1.6 to localize to the AIS, a different mechanism is responsible for the localization and membrane dynamics in the somatodendritic region of hippocampal neurons.