Search

Membrane contact sites between endoplasmic reticulum (ER) and plasma membrane (PM), or ER-PM junctions, are found in all eukaryotic cells. In excitable cells they play unique roles in organizing diverse forms of Ca2+ signaling as triggered by membrane depolarization. ER-PM junctions underlie crucial physiological processes such as excitation-contraction coupling, smooth muscle contraction and relaxation, and various forms of activity-dependent signaling and plasticity in neurons. In many cases the structure and molecular composition of ER-PM junctions in excitable cells comprise important regulatory feedback loops linking depolarization-induced Ca2+ signaling at these sites to the regulation of membrane potential. Here, we describe recent findings on physiological roles and molecular composition of native ER-PM junctions in excitable cells. We focus on recent studies that provide new insights into canonical forms of depolarization-induced Ca2+ signaling occurring at junctional triads and dyads of striated muscle, as well as the diversity of ER-PM junctions in these cells and in smooth muscle and neurons.

The 6TM family of K channels comprises the voltage-gated KV subfamilies, the EAG subfamily (which includes hERG channels), the Ca2+-activated Slo subfamily (actually with 7TM, termed BK) and the Ca2+-activated SK subfamily. These channels possess a pore-forming α subunit that comprise tetramers of identical subunits (homomeric) or of different subunits (heteromeric). Heteromeric channels can only be formed within subfamilies (e.g. Kv1.1 with Kv1.2; Kv7.2 with Kv7.3). The pharmacology largely reflects the subunit composition of the functional channel.

Mapping neuronal networks that underlie behavior has become a central focus in neuroscience. While serial section electron microscopy (ssEM) can reveal the fine structure of neuronal networks (connectomics), it does not provide the molecular information that helps identify cell types or their functional properties. Volumetric correlated light and electron microscopy (vCLEM) combines ssEM and volumetric fluorescence microscopy to incorporate molecular labeling into ssEM datasets. We developed an approach that uses small fluorescent single-chain variable fragment (scFv) immuno-probes to perform multiplexed detergent-free immuno-labeling and serial electron microscopy on the same samples. We generated eight such fluorescent scFvs that targeted useful markers for brain studies (GFP, GFAP, calbindin, parvalbumin, Kv1.2, VGluT1, PSD-95, and neuropeptide Y). To test the vCLEM approach, six different fluorescent probes were imaged in a sample of the cortex of a cerebellar lobule (Crus 1), using confocal microscopy with linear unmixing, followed by ssEM imaging of the same sample. The results show excellent ultrastructure and superimposition of the different fluorescence channels. We document a poorly described cell type in the cerebellum, two types of mossy fiber terminals, and the subcellular localization of ion channels. Because scFvs can be derived from existing monoclonal antibodies, hundreds of such probes can be generated to enable a wide range of connectomic studies.

The 6TM family of K channels comprises the voltage-gated KV subfamilies, the EAG subfamily (which includes hERG channels), the Ca2+-activated Slo subfamily (actually with 7TM, termed BK) and the Ca2+-activated SK subfamily. These channels possess a pore-forming α subunit that comprise tetramers of identical subunits (homomeric) or of different subunits (heteromeric). Heteromeric channels can only be formed within subfamilies (e.g. Kv1.1 with Kv1.2; Kv7.2 with Kv7.3). The pharmacology largely reflects the subunit composition of the functional channel.Kv7 channelsKv7.1-Kv7.5 (KCNQ1-5) K+ channels are voltage-gated K+ channels with major roles in neurons, muscle cells and epithelia where they underlie physiologically important K+ currents, such as the neuronal M-current and the cardiac IKs. Genetic deficiencies in all five KCNQ genes result in human excitability disorders, including epilepsy, autism spectrum disorders, cardiac arrhythmias and deafness. Thanks to the recent knowledge of the structure and function of human KCNQ-encoded proteins, these channels are increasingly used as drug targets for treating diseases [326, 2, 767].

Membrane contact sites between endoplasmic reticulum (ER) and plasma membrane (PM), or ER-PM junctions, are found in all eukaryotic cells. In excitable cells they play unique roles in organizing diverse forms of Ca

Basic neuroscience research employs numerous forms of antibodies as key reagents in diverse applications. While the predominant use of antibodies is as immunolabeling reagents, neuroscientists are making increased use of intracellular antibodies or intrabodies. Intrabodies are recombinant antibodies genetically encoded for expression within neurons. These can be used to target various cargo (fluorescent proteins, reporters, enzymes, etc.) to specific molecules and subcellular domains to report on and manipulate neuronal function with high precision. Intrabodies have the advantages inherent in all genetically encoded recombinant antibodies but represent a distinct subclass in that their structure allows for their expression and function within cells. The high precision afforded by the ability to direct their expression to specific cell types, and the selective binding of intrabodies to targets within these allows intrabodies to offer unique advantages for neuroscience research, given the tremendous molecular, cellular and morphological complexity of brain neurons. Intrabodies expressed within neurons have been used for a variety of purposes in basic neuroscience research. Here I provide a general background to intrabodies and their development, and examples of their emerging utility as valuable basic neuroscience research tools.

Selective ion channel modulators play a critical role in physiology in defining the contribution of specific ion channels to physiological function and as proof of concept for novel therapeutic strategies. Antibodies are valuable research tools that have broad uses including defining the expression and localization of ion channels in native tissue, and capturing ion channel proteins for subsequent analyses. In this review, we detail how renewable and recombinant antibodies can be used to control ion channel function. We describe the different forms of renewable and recombinant antibodies that have been used and the mechanisms by which they modulate ion channel function. We highlight the use of recombinant antibodies that are expressed intracellularly (intrabodies) as genetically-encoded tools to control ion channel function. We also offer perspectives of avenues of future research that may be opened by the application of emerging technologies for engineering recombinant antibodies for enhanced utility in ion channel research. Overall, this review provides insights that may help stimulate and guide interested researchers to develop and incorporate renewable and recombinant antibodies as valuable tools to control ion channel function.

Significance Our data challenge the generally accepted view that Kv2.1 proteins regulate arterial smooth muscle function by regulating their membrane potential. Rather, we discovered that Kv2.1 plays both conductive and structural roles with opposing functional consequences on arterial myocytes, with the former predominating in males, the latter in females. Opening of Kv2.1 channels opposes vasoconstriction by inducing membrane hyperpolarization. In addition to this conductive function, Kv2.1 promotes the structural clustering of CaV1.2 channels, thereby enhancing Ca2+ influx and inducing vasoconstriction. These two functions are highlighted by differences in the regulation of membrane potential, intracellular Ca2+, and myogenic tone between males and females. Our data suggest that these disparities derive from sex-specific variations in Kv2.1 expression levels in male versus female myocytes., The accepted role of the protein Kv2.1 in arterial smooth muscle cells is to form K+ channels in the sarcolemma. Opening of Kv2.1 channels causes membrane hyperpolarization, which decreases the activity of L-type CaV1.2 channels, lowering intracellular Ca2+ ([Ca2+]i) and causing smooth muscle relaxation. A limitation of this model is that it is based exclusively on data from male arterial myocytes. Here, we used a combination of electrophysiology as well as imaging approaches to investigate the role of Kv2.1 channels in male and female arterial myocytes. We confirmed that Kv2.1 plays a canonical conductive role but found it also has a structural role in arterial myocytes to enhance clustering of CaV1.2 channels. Less than 1% of Kv2.1 channels are conductive and induce membrane hyperpolarization. Paradoxically, by enhancing the structural clustering and probability of CaV1.2–CaV1.2 interactions within these clusters, Kv2.1 increases Ca2+ influx. These functional impacts of Kv2.1 depend on its level of expression, which varies with sex. In female myocytes, where expression of Kv2.1 protein is higher than in male myocytes, Kv2.1 has conductive and structural roles. Female myocytes have larger CaV1.2 clusters, larger [Ca2+]i, and larger myogenic tone than male myocytes. In contrast, in male myocytes, Kv2.1 channels regulate membrane potential but not CaV1.2 channel clustering. We propose a model in which Kv2.1 function varies with sex: in males, Kv2.1 channels control membrane potential but, in female myocytes, Kv2.1 plays dual electrical and CaV1.2 clustering roles. This contributes to sex-specific regulation of excitability, [Ca2+]i, and myogenic tone in arterial myocytes.

In mammalian brain neurons, membrane depolarization leads to voltage-gated Ca2+ channel-mediated Ca2+ influx that triggers diverse cellular responses, including gene expression, in a process termed excitation–transcription coupling. Neuronal L-type Ca2+ channels, which have prominent populations on the soma and distal dendrites of hippocampal neurons, play a privileged role in excitation–transcription coupling. The voltage-gated K+ channel Kv2.1 organizes signaling complexes containing the L-type Ca2+ channel Cav1.2 at somatic endoplasmic reticulum–plasma membrane junctions. This leads to enhanced clustering of Cav1.2 channels, increasing their activity. However, the downstream consequences of the Kv2.1-mediated regulation of Cav1.2 localization and function on excitation–transcription coupling are not known. Here, we have identified a region between residues 478 to 486 of Kv2.1’s C terminus that mediates the Kv2.1-dependent clustering of Cav1.2. By disrupting this Ca2+ channel association domain with either mutations or with a cell-penetrating interfering peptide, we blocked the Kv2.1-mediated clustering of Cav1.2 at endoplasmic reticulum–plasma membrane junctions and the subsequent enhancement of its channel activity and somatic Ca2+ signals without affecting the clustering of Kv2.1. These interventions abolished the depolarization-induced and L-type Ca2+ channel-dependent phosphorylation of the transcription factor CREB and the subsequent expression of c-Fos in hippocampal neurons. Our findings support a model whereby the Kv2.1-Ca2+ channel association domain-mediated clustering of Cav1.2 channels imparts a mechanism to control somatic Ca2+ signals that couple neuronal excitation to gene expression.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15539. Ion channels are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate. ispartof: BRITISH JOURNAL OF PHARMACOLOGY vol:178 pages:S157-S245 ispartof: location:England status: published

Significance In hippocampal neurons, gene expression is triggered by electrical activity and Ca2+ entry via L-type Cav1.2 channels in a process called excitation–transcription coupling. We identified a domain on the voltage-gated K+ channel Kv2.1 that promotes the clustering of L-type Cav1.2 channels at endoplasmic reticulum–plasma membrane junctions in the soma of neurons. Importantly, we discovered by disrupting this domain that the Kv2.1-mediated clustering of Cav1.2 at this somatic microdomain is critical for depolarization-induced excitation–transcription coupling., In mammalian brain neurons, membrane depolarization leads to voltage-gated Ca2+ channel-mediated Ca2+ influx that triggers diverse cellular responses, including gene expression, in a process termed excitation–transcription coupling. Neuronal L-type Ca2+ channels, which have prominent populations on the soma and distal dendrites of hippocampal neurons, play a privileged role in excitation–transcription coupling. The voltage-gated K+ channel Kv2.1 organizes signaling complexes containing the L-type Ca2+ channel Cav1.2 at somatic endoplasmic reticulum–plasma membrane junctions. This leads to enhanced clustering of Cav1.2 channels, increasing their activity. However, the downstream consequences of the Kv2.1-mediated regulation of Cav1.2 localization and function on excitation–transcription coupling are not known. Here, we have identified a region between residues 478 to 486 of Kv2.1’s C terminus that mediates the Kv2.1-dependent clustering of Cav1.2. By disrupting this Ca2+ channel association domain with either mutations or with a cell-penetrating interfering peptide, we blocked the Kv2.1-mediated clustering of Cav1.2 at endoplasmic reticulum–plasma membrane junctions and the subsequent enhancement of its channel activity and somatic Ca2+ signals without affecting the clustering of Kv2.1. These interventions abolished the depolarization-induced and L-type Ca2+ channel-dependent phosphorylation of the transcription factor CREB and the subsequent expression of c-Fos in hippocampal neurons. Our findings support a model whereby the Kv2.1-Ca2+ channel association domain-mediated clustering of Cav1.2 channels imparts a mechanism to control somatic Ca2+ signals that couple neuronal excitation to gene expression.

The 6TM family of K channels comprises the voltage-gated KV subfamilies, the EAG subfamily (which includes hERG channels), the Ca2+-activated Slo subfamily (actually with 7TM, termed BK) and the Ca2+-activated SK subfamily. These channels possess a pore-forming α subunit that comprise tetramers of identical subunits (homomeric) or of different subunits (heteromeric). Heteromeric channels can only be formed within subfamilies (e.g. Kv1.1 with Kv1.2; Kv7.2 with Kv7.3). The pharmacology largely reflects the subunit composition of the functional channel.

Ion channels are often found in dense clusters within the plasma membranes of excitable cells. Based on experimental measurements of a wide range of channels in various cell types, Sato et al. propose that channel clusters form stochastically and that their size is regulated by a common feedback mechanism., Ion channels are often found arranged into dense clusters in the plasma membranes of excitable cells, but the mechanisms underlying the formation and maintenance of these functional aggregates are unknown. Here, we tested the hypothesis that channel clustering is the consequence of a stochastic self-assembly process and propose a model by which channel clusters are formed and regulated in size. Our hypothesis is based on statistical analyses of the size distributions of the channel clusters we measured in neurons, ventricular myocytes, arterial smooth muscle, and heterologous cells, which in all cases were described by exponential functions, indicative of a Poisson process (i.e., clusters form in a continuous, independent, and memory-less fashion). We were able to reproduce the observed cluster distributions of five different types of channels in the membrane of excitable and tsA-201 cells in simulations using a computer model in which channels are “delivered” to the membrane at randomly assigned locations. The model’s three parameters represent channel cluster nucleation, growth, and removal probabilities, the values of which were estimated based on our experimental measurements. We also determined the time course of cluster formation and membrane dwell time for CaV1.2 and TRPV4 channels expressed in tsA-201 cells to constrain our model. In addition, we elaborated a more complex version of our model that incorporated a self-regulating feedback mechanism to shape channel cluster formation. The strong inference we make from our results is that CaV1.2, CaV1.3, BK, and TRPV4 proteins are all randomly inserted into the plasma membranes of excitable cells and that they form homogeneous clusters that increase in size until they reach a steady state. Further, it appears likely that cluster size for a diverse set of membrane-bound proteins and a wide range of cell types is regulated by a common feedback mechanism.

Fortenbach et al. use genetic deletion of the K+ channel Kv2.1 to quantitatively dissect the ionic mechanisms contributing to the dark current of vertebrate rod photoreceptors. Loss of Kv2.1 resulted in reduced dark current, depolarization, increased intracellular free Ca2+, disruption of normal inner segment structure, and progressive photoreceptor degeneration., Vertebrate retinal photoreceptors signal light by suppressing a circulating “dark current” that maintains their relative depolarization in the dark. This dark current is composed of an inward current through CNG channels and NCKX transporters in the outer segment that is balanced by outward current exiting principally from the inner segment. It has been hypothesized that Kv2.1 channels carry a predominant fraction of the outward current in rods. We examined this hypothesis by comparing whole cell, suction electrode, and electroretinographic recordings from Kv2.1 knockout (Kv2.1−/−) and wild-type (WT) mouse rods. Single cell recordings revealed flash responses with unusual kinetics, and reduced dark currents that were quantitatively consistent with the measured depolarization of the membrane resting potential in the dark. A two-compartment (outer and inner segment) physiological model based on known ionic mechanisms revealed that the abnormal Kv2.1−/− rod photoresponses arise principally from the voltage dependencies of the known conductances and the NCKX exchanger, and a highly elevated fraction of inward current carried by Ca2+ through CNG channels due to the aberrant depolarization. Kv2.1−/− rods had shorter outer segments than WT and dysmorphic mitochondria in their inner segments. Optical coherence tomography of knockout animals demonstrated a slow photoreceptor degeneration over a period of 6 mo. Overall, these findings reveal that Kv2.1 channels carry 70–80% of the non-NKX outward dark current of the mouse rod, and that the depolarization caused by the loss of Kv2.1 results in elevated Ca2+ influx through CNG channels and elevated free intracellular Ca2+, leading to progressive degeneration.

Nanobodies (nAbs) are recombinant antigen-binding variable domain fragments obtained from heavy-chain-only immunoglobulins. Among mammals, these are unique to camelids (camels, llamas, alpacas, etc.). Nanobodies are of great use in biomedical research due to their efficient folding and stability under a variety of conditions, as well as their small size. The latter characteristic is particularly important for nAbs used as immunolabeling reagents, since this can improve penetration of cell and tissue samples compared to conventional antibodies, and also reduce the gap distance between signal and target, thereby improving imaging resolution. In addition, their recombinant nature allows for unambiguous definition and permanent archiving in the form of DNA sequence, enhanced distribution in the form of sequences or plasmids, and easy and inexpensive production using well-established bacterial expression systems, such as the IPTG induction method described here. This article will review the basic workflow and process for developing, screening, and validating novel nAbs against neuronal target proteins. The protocols described make use of the most common nAb development method, wherein an immune repertoire from an immunized llama is screened via phage display technology. Selected nAbs can then be taken through validation assays for use as immunolabels or as intrabodies in neurons. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Total RNA isolation from camelid leukocytes Basic Protocol 2: First-strand cDNA synthesis; VH H and VH repertoire PCR Basic Protocol 3: Preparation of the phage display library Basic Protocol 4: Panning of the phage display library Basic Protocol 5: Small-scale nAb expression Basic Protocol 6: Sequence analysis of selected nAb clones Basic Protocol 7: Nanobody validation as immunolabels Basic Protocol 8: Generation of nAb-pEGFP mammalian expression constructs Basic Protocol 9: Nanobody validation as intrabodies Support Protocol 1: ELISA for llama serum testing, phage titer, and screening of selected clones Support Protocol 2: Amplification of helper phage stock Support Protocol 3: nAb expression in amber suppressor E. coli bacterial strains.

Basic neuroscience research employs antibodies as key reagents to label, capture, and modulate the function of proteins of interest. Antibodies are immunoglobulin proteins. Recombinant antibodies are immunoglobulin proteins whose nucleic acid coding regions, or fragments thereof, have been cloned into expression plasmids that allow for unlimited production. Recombinant antibodies offer many advantages over conventional antibodies including their unambiguous identification and digital archiving via DNA sequencing, reliable expression, ease and reliable distribution as DNA sequences and as plasmids, and the opportunity for numerous forms of engineering to enhance their utility. Recombinant antibodies exist in many different forms, each of which offers potential advantages and disadvantages for neuroscience research applications. I provide an overview of recombinant antibodies and their development. Examples of their emerging use as valuable reagents in basic neuroscience research are also discussed. Many of these examples employ recombinant antibodies in innovative experimental approaches that cannot be pursued with conventional antibodies. © 2020 Wiley Periodicals LLC.

Methods for one-photon fluorescent imaging of calcium dynamics in vivo are popular due to their ability to simultaneously capture the dynamics of hundreds of neurons across large fields of view, at a low equipment complexity and cost. In contrast to two-photon methods, however, one-photon methods suffer from higher levels of crosstalk between cell bodies and the surrounding neuropil, resulting in decreased signal-to-noise and artifactual correlations of neural activity. Here, we address this problem by engineering cell body-targeted variants of the fluorescent calcium indicator GCaMP6f. We screened fusions of GCaMP6f to both natural as well as engineered peptides, and identified fusions that localized GCaMP6f to within approximately 50 microns of the cell body of neurons in live mice and larval zebrafish. One-photon imaging of soma-targeted GCaMP6f in dense neural circuits reported fewer artifactual spikes from neuropil, increased signal-to-noise ratio, and decreased artifactual correlation across neurons. Thus, soma-targeting of fluorescent calcium indicators increases neuronal signal fidelity and may facilitate even greater usage of simple, powerful, one-photon methods of population imaging of neural calcium dynamics.

In the wake of the reproducibility crisis and numerous discussions on how commercially available antibodies as research tool contribute to it, The Antibody Society developed a series of 10 webinars to address the issues involved. The webinars were delivered by speakers with both academic and commercial backgrounds. This report highlights the problems, and offers solutions to help the scientific community appropriately identify the right antibodies and to validate them for their research and development projects. Despite the various solutions proposed here, they must be applied on a case-by-case basis. Each antibody must be verified based on the content of the product sheet, and subsequently through experimentation to confirm integrity, specificity and selectivity. Verification needs to focus on the precise application and tissue/cell type for which the antibody will be used, and all verification data must be reported openly. The various approaches discussed here all have caveats, so a combination of solutions must be considered.

The association of plasma membrane (PM)-localized voltage-gated potassium (Kv2) channels with endoplasmic reticulum (ER)-localized vesicle-associated membrane protein-associated proteins VAPA and VAPB defines ER–PM junctions in mammalian brain neurons. Here, we used proteomics to identify proteins associated with Kv2/VAP-containing ER–PM junctions. We found that the VAP-interacting membrane-associated phosphatidylinositol (PtdIns) transfer proteins PYK2 N-terminal domain-interacting receptor 2 (Nir2) and Nir3 specifically associate with Kv2.1 complexes. When coexpressed with Kv2.1 and VAPA in HEK293T cells, Nir2 colocalized with cell-surface–conducting and -nonconducting Kv2.1 isoforms. This was enhanced by muscarinic-mediated PtdIns(4,5)P(2) hydrolysis, leading to dynamic recruitment of Nir2 to Kv2.1 clusters. In cultured rat hippocampal neurons, exogenously expressed Nir2 did not strongly colocalize with Kv2.1, unless exogenous VAPA was also expressed, supporting the notion that VAPA mediates the spatial association of Kv2.1 and Nir2. Immunolabeling signals of endogenous Kv2.1, Nir2, and VAP puncta were spatially correlated in cultured neurons. Fluorescence-recovery-after-photobleaching experiments revealed that Kv2.1, VAPA, and Nir2 have comparable turnover rates at ER–PM junctions, suggesting that they form complexes at these sites. Exogenous Kv2.1 expression in HEK293T cells resulted in significant differences in the kinetics of PtdIns(4,5)P(2) recovery following repetitive muscarinic stimulation, with no apparent impact on resting PtdIns(4,5)P(2) or PtdIns(4)P levels. Finally, the brains of Kv2.1-knockout mice had altered composition of PtdIns lipids, suggesting a crucial role for native Kv2.1-containing ER–PM junctions in regulating PtdIns lipid metabolism in brain neurons. These results suggest that ER–PM junctions formed by Kv2 channel–VAP pairing regulate PtdIns lipid homeostasis via VAP-associated PtdIns transfer proteins.

The voltage-gated K+ channel Kv2.1 serves a major structural role in the soma and proximal dendrites of mammalian brain neurons, tethering the plasma membrane (PM) to endoplasmic reticulum (ER). Although Kv2.1 clustering at neuronal ER-PM junctions (EPJs) is tightly regulated and highly conserved, its function remains unclear. By identifying and evaluating proteins in close spatial proximity to Kv2.1-containing EPJs, we discovered that a significant role of Kv2.1 at EPJs is to promote the clustering and functional coupling of PM L-type Ca2+ channels (LTCCs) to ryanodine receptor (RyR) ER Ca2+ release channels. Kv2.1 clustering also unexpectedly enhanced LTCC opening at polarized membrane potentials. This enabled Kv2.1-LTCC-RyR triads to generate localized Ca2+ release events (i.e., Ca2+ sparks) independently of action potentials. Together, these findings uncover a novel mode of LTCC regulation and establish a unique mechanism whereby Kv2.1-associated EPJs provide a molecular platform for localized somatodendritic Ca2+ signals in mammalian brain neurons.

Kv2.1 is a major delayed-rectifier voltage-gated potassium channel widely expressed in neurons of the CNS. Kv2.1 localizes in high-density cell-surface clusters in the soma and proximal dendrites as well as in the axon initial segment (AIS). Given the crucial roles of both of these compartments in integrating signal input and then generating output, this localization of Kv2.1 is ideal for regulating the overall excitability of neurons. Here we used fluorescence recovery after photobleaching imaging, mutagenesis, and pharmacological interventions to investigate the molecular mechanisms that control the localization of Kv2.1 in these two different membrane compartments in cultured rat hippocampal neurons of mixed sex. Our data uncover a unique ability of Kv2.1 channels to use two molecularly distinct trafficking pathways to accomplish this. Somatodendritic Kv2.1 channels are targeted by the conventional secretory pathway, whereas axonal Kv2.1 channels are targeted by a nonconventional trafficking pathway independent of the Golgi apparatus. We further identified a new AIS trafficking motif in the C-terminus of Kv2.1, and show that putative phosphorylation sites in this region are critical for the restricted and clustered localization in the AIS. These results indicate that neurons can regulate the expression and clustering of Kv2.1 in different membrane domains independently by using two distinct localization mechanisms, which would allow neurons to precisely control local membrane excitability.SIGNIFICANCE STATEMENTOur study uncovered a novel mechanism that targets the Kv2.1 voltage-gated potassium channel to two distinct trafficking pathways and two distinct subcellular destinations: the somatodendritic plasma membrane and that of the axon initial segment. We also identified a distinct motif, including putative phosphorylation sites, that is important for the AIS localization. This raises the possibility that the destination of a channel protein can be dynamically regulated via changes in post-translational modification, which would impact the excitability of specific membrane compartments.

Developmental and epileptic encephalopathies (DEE) are a group of severe epilepsies that usually present with intractable seizures, developmental delay and are at a higher risk for premature mortality. Numerous genes have been identified as a monogenic cause of DEE, includingKCNB1. The voltage-gated potassium channel KV2.1, encoded byKCNB1, is primarily responsible for delayed rectifier potassium currents that are important regulators of excitability in electrically excitable cells, including neurons and cardiomyocytes. Thede novopathogenic variantKCNB1-p.G379R was identified in an infant with epileptic spasms, atonic, focal and tonic-clonic seizures that were refractory to treatment with standard antiepileptic drugs. Previous work demonstrated deficits in potassium conductance, but did not assess non-conducting functions. To determine if the G379R variant affected clustering at endoplasmic reticulum-plasma membrane junctions KV2.1-G379R was expressed in HEK293T cells. KV2.1-G379R expression did not induce formation of endoplasmic reticulum-plasma membrane junctions, and co-expression of KV2.1-G379R with KV2.1-WT lowered induction of these structures relative to KV2.1-WT alone, suggesting a dominant negative effect. To model this variantin vivo, we introducedKcnb1G379Rinto mice using CRISPR/Cas9 genome editing. We characterized neurological and neurobehavioral phenotypes ofKcnb1G379R/+(Kcnb1R/+) andKcnb1G379R/G379R(Kcnb1R/R) mice, and screened for cardiac abnormalities. Immunohistochemistry studies on brains fromKcnb1+/+(WT), Kcnb1R/+andKcnb1R/Rmice revealed genotype-dependent differences in the levels and subcellular localization of KV2.1, with reduced plasma membrane expression of the KV2.1-G379R protein, consistent within vitrodata.Kcnb1R/+andKcnb1R/Rmice displayed profound hyperactivity, repetitive behaviors, impulsivity and reduced anxiety. In addition, bothKcnb1R/+andKcnb1R/Rmice exhibited abnormal interictal EEG abnormalities, including isolated spike and slow waves. Spontaneous seizure events were observed inKcnb1R/Rmice during exposure to novel environments and/or handling, while bothKcnb1R/+andKcnb1R/Rmutants were more susceptible to induced seizures.Kcnb1R/+andKcnb1R/Rmice exhibited prolonged rate-corrected QT interval on surface ECG recording. Overall, theKcnb1G379Rmice recapitulate many features observed in individuals with DEE due to pathogenic variants inKCNB1. This new mouse model ofKCNB1associated DEE will be valuable for improving the understanding of the underlying pathophysiology and will provide a valuable tool for the development of therapies to treat this pharmacoresistant DEE.

Nanobodies (nAbs) are small, minimal antibodies that have distinct attributes that make them uniquely suited for certain biomedical research, diagnostic and therapeutic applications. Prominent uses include as intracellular antibodies or intrabodies to bind and deliver cargo to specific proteins and/or subcellular sites within cells, and as nanoscale immunolabels for enhanced tissue penetration and improved spatial imaging resolution. Here, we report the generation and validation of nAbs against a set of proteins prominently expressed at specific subcellular sites in mammalian brain neurons. We describe a novel hierarchical validation pipeline to systematically evaluate nAbs isolated by phage display for effective and specific use as intrabodies and immunolabels in mammalian cells including brain neurons. These nAbs form part of a robust toolbox for targeting proteins with distinct and highly spatially-restricted subcellular localization in mammalian brain neurons, allowing for visualization and/or modulation of structure and function at those sites.

The voltage-gated K+channel Kv2.1 serves a major structural role in the soma and proximal dendrites of brain neurons, tethering the plasma membrane (PM) to the endoplasmic reticulum (ER). Although Kv2.1 clustering at neuronal ER-PM junctions (EPJs) is tightly regulated and conserved across species, its function at these sites is unclear. By identifying and evaluating proteins in close spatial proximity to Kv2.1-containing EPJs, we discovered that a significant role of Kv2.1 at EPJs is to promote the clustering and functional coupling of PM L-type Ca2+channels (LTCCs) to ryanodine receptor (RyR) ER Ca2+release channels. Kv2.1 clustering also unexpectedly enhanced LTCC opening at polarized membrane potentials. This enabled Kv2.1-LTCC-RyR triads to generate localized Ca2+release events (i.e., Ca2+sparks) independently of action potentials. Together, these findings uncover a novel mode of LTCC regulation and establish a unique mechanism whereby Kv2.1-associated EPJs provide a molecular platform for localized somatodendritic Ca2+signals.

Temporal lobe epilepsy (TLE) is a prevalent neurological disorder with many patients experiencing poor seizure control with existing anti-epileptic drugs. Thus, novel insights into the mechanisms of epileptogenesis and identification of new drug targets can be transformative. Changes in ion channel function have been shown to play a role in generating the aberrant neuronal activity observed in TLE. Previous work demonstrates that hyperpolarization-activated cyclic nucleotide-gated (HCN) channels regulate neuronal excitability and are mislocalized within CA1 pyramidal cells in a rodent model of TLE. The subcellular distribution of HCN channels is regulated by an auxiliary subunit, tetratricopeptide repeat–containing Rab8b-interacting protein (TRIP8b), and disruption of this interaction correlates with channel mislocalization. However, the molecular mechanisms responsible for HCN channel dysregulation in TLE are unclear. Here we investigated whether changes in TRIP8b phosphorylation are sufficient to alter HCN channel function. We identified a phosphorylation site at residue Ser(237) of TRIP8b that enhances binding to HCN channels and influences channel gating by altering the affinity of TRIP8b for the HCN cytoplasmic domain. Using a phosphospecific antibody, we demonstrate that TRIP8b phosphorylated at Ser(237) is enriched in CA1 distal dendrites and that phosphorylation is reduced in the kainic acid model of TLE. Overall, our findings indicate that the TRIP8b–HCN interaction can be modulated by changes in phosphorylation and suggest that loss of TRIP8b phosphorylation may affect HCN channel properties during epileptogenesis. These results highlight the potential of drugs targeting posttranslational modifications to restore TRIP8b phosphorylation to reduce excitability in TLE.

The overarching goal of the NIH BRAIN (Brain Research through Advancing Innovative Neurotechnologies) Initiative is to advance the understanding of healthy and diseased brain circuit function through technological innovation. Core principles for this goal include the validation and dissemination of the myriad innovative technologies, tools, methods, and resources emerging from BRAIN-funded research. Innovators, BRAIN funding agencies, and non-Federal partners are working together to develop strategies for making these products usable, available, and accessible to the scientific community. Here, we describe several early strategies for supporting the dissemination of BRAIN technologies. We aim to invigorate a dialogue with the neuroscience research and funding community, interdisciplinary collaborators, and trainees about the existing and future opportunities for cultivating groundbreaking research products into mature, integrated, and adaptable research systems. Along with the accompanying Society for Neuroscience 2019 Mini-Symposium, “BRAIN Initiative: Cutting-Edge Tools and Resources for the Community,” we spotlight the work of several BRAIN investigator teams who are making progress toward providing tools, technologies, and services for the neuroscience community. These tools access neural circuits at multiple levels of analysis, from subcellular composition to brain-wide network connectivity, including the following: integrated systems for EM- and florescence-based connectomics, advances in immunolabeling capabilities, and resources for recording and analyzing functional connectivity. Investigators describe how the resources they provide to the community will contribute to achieving the goals of the NIH BRAIN Initiative. Finally, in addition to celebrating the contributions of these BRAIN-funded investigators, the Mini-Symposium will illustrate the broader diversity of BRAIN Initiative investments in cutting-edge technologies and resources.

Transient receptor potential melastatin 4 (TRPM4) is a Ca2+ -activated nonselective cationic channel that regulates cell migration and contractility. Increased TRPM4 expression has been related to pathologies, in which cytoskeletal rearrangement and cell migration are altered, such as metastatic cancer. Here, we identify the K+ channel tetramerization domain 5 (KCTD5) protein, a putative adaptor of cullin3 E3 ubiquitin ligase, as a novel TRPM4-interacting protein. We demonstrate that KCTD5 is a positive regulator of TRPM4 activity by enhancing its Ca2+ sensitivity. We show that through its effects on TRPM4 that KCTD5 promotes cell migration and contractility. Finally, we observed that both TRPM4 and KCTD5 expression are increased in distinct patterns in different classes of breast cancer tumor samples. Together, these data support that TRPM4 activity can be regulated through expression levels of either TRPM4 or KCTD5, not only contributing to increased understanding of the molecular mechanisms involved on the regulation of these important ion channels, but also providing information that could inform treatments based on targeting these distinct molecules that define TRPM4 activity.

SUMMARYNanobodies (nAbs) are small, minimal antibodies that have distinct attributes that make them uniquely suited for certain biomedical research, diagnostic and therapeutic applications. Prominent uses include as intracellular antibodies or intrabodies to bind and deliver cargo to specific proteins and/or subcellular sites within cells, and as nanoscale immunolabels for enhanced tissue penetration and improved spatial imaging resolution. Here, we report the generation and validation of nAbs against a set of proteins prominently expressed at specific subcellular sites in brain neurons. We describe a novel hierarchical validation pipeline to systematically evaluate nAbs isolated by phage display for effective and specific use as intrabodies and immunolabels in mammalian cells including brain neurons. These nAbs form part of a robust toolbox for targeting proteins with distinct and highly spatially-restricted subcellular localization in mammalian brain neurons, allowing for visualization and/or modulation of structure and function at those sites.

Voltage-dependent K+ (potassium; Kv) channels are ion channels that critically impact neuronal excitability and function. Four principal α subunits assemble to create a membrane-spanning pore that opens in a voltage-dependent manner to allow the selective passage of K+ ions across the cell membrane. Forty human genes encoding Kv channel α subunits have been identified, and most of them are expressed in the nervous system. The individual Kv subunits display unique cellular and subcellular expression patterns and co-assemble into distinct homo- and hetero-tetrameric channels that differ in their electrophysiological and pharmacological properties, and their sensitivity to dynamic modulation, by cellular signaling pathways. The resulting diversity allows Kv channels to impact all steps in electrical information processing, as well as numerous other aspects of neuronal functions, including those in which they appear to play a non-conducting role. This chapter reviews the current basic knowledge about this large and important family of ion channels.

Generating recombinant monoclonal antibodies (R-mAbs) from mAb-producing hybridomas offers numerous advantages that increase the effectiveness, reproducibility, and transparent reporting of research. We report here the generation of a novel resource in the form of a library of recombinant R-mAbs validated for neuroscience research. We cloned immunoglobulin G (IgG) variable domains from cryopreserved hybridoma cells and input them into an integrated pipeline for expression and validation of functional R-mAbs. To improve efficiency over standard protocols, we eliminated aberrant Sp2/0-Ag14 hybridoma-derived variable light transcripts using restriction enzyme treatment. Further, we engineered a plasmid backbone that allows for switching of the IgG subclasses without altering target binding specificity to generate R-mAbs useful in simultaneous multiplex labeling experiments not previously possible. The method was also employed to rescue IgG variable sequences and generate functional R-mAbs from a non-viable cryopreserved hybridoma. All R-mAb sequences and plasmids will be archived and disseminated from open source suppliers., eLife digest The immune system fights off disease-causing microbes using antibodies: Y-shaped proteins that each bind to a specific foreign molecule. Indeed, these proteins bind so tightly and so specifically that they can pick out a single target in a complex mixture of different molecules. This property also makes them useful in research. For example, neurobiologists can use antibodies to mark target proteins in thin sections of brain tissue. This reveals their position inside brain cells, helping to link the structure of the brain to the roles the different parts of this structure perform. To use antibodies in this way, scientists need to be able to produce them in large quantities without losing their target specificity. The most common way to do this is with cells called hybridomas. A hybridoma is a hybrid of an antibody-producing immune cell and a cancer cell, and it has properties of both. From the immune cell, it inherits the genes to make a specific type of antibody. From the cancer cell, it inherits the ability to go on dividing forever. In theory, hybridomas should be immortal antibody factories, but they have some limitations. They are expensive to keep alive, hard to transport between labs, and their genes can be unstable. Problems can creep into their genetic code, halting their growth or changing the targets their antibodies recognize. When this happens, scientists can lose vital research tools. Instead of keeping the immune cells alive, an alternative approach is to make recombinant antibodies. Rather than store the whole cell, this approach just stores the parts of the genes that encode antibody target-specificity. Andrews et al. set out to convert a valuable toolbox of neuroscience antibodies into recombinant form. This involved copying the antibody genes from a large library of preserved hybridoma cells. However, many hybridomas also carry genes that produce non-functional antibodies. A step in the process removed these DNA sequences, ensuring that only working antibodies made it into the final library. Using frozen cells made it possible to recover antibody genes from hybridoma cells that could no longer grow. The recombinant DNA sequences provide a permanent record of useful antibodies. Not only does this prevent the loss of research tools, it is also much more shareable than living cells. Modifications to the DNA sequences in the library allow for the use of many antibodies at once. This could help when studying the interactions between different molecules in the brain. Toolkits like these could also make it easier to collaborate, and to reproduce data gathered by different researchers around the world.

Summary Dorsal root ganglion (DRG) sensory neuron subtypes defined by their in vivo properties display distinct intrinsic electrical properties. We used bulk RNA sequencing of genetically labeled neurons and electrophysiological analyses to define ion channel contributions to the intrinsic electrical properties of DRG neuron subtypes. The transcriptome profiles of eight DRG neuron subtypes revealed differentially expressed and functionally relevant genes, including voltage-gated ion channels. Guided by these data, electrophysiological analyses using pharmacological and genetic manipulations as well as computational modeling of DRG neuron subtypes were undertaken to assess the functions of select voltage-gated potassium channels (Kv1, Kv2, Kv3, and Kv4) in shaping action potential (AP) waveforms and firing patterns. Our findings show that the transcriptome profiles have predictive value for defining ion channel contributions to sensory neuron subtype-specific intrinsic physiological properties. The distinct ensembles of voltage-gated ion channels predicted to underlie the unique intrinsic physiological properties of eight DRG neuron subtypes are presented.