Antony P. Jackson

The sodium channel β‐subunit SCN3b modulates the kinetics of SCN5a and is expressed heterogeneously in sheep heart

1 Cardiac sodium channels are composed of a pore‐forming α‐subunit, SCN5a, and one or more auxiliary β‐subunits. The aim of this study was to investigate the role of the recently discovered member of the β‐subunit family, SCN3b, in the heart. 2 Northern blot and Western blot studies show that SCN3b is highly expressed in the ventricles and Purkinje fibres but not in atrial tissue. This is in contrast to the uniform expression of SCN1b throughout the heart. 3 Co‐expression of SCN3b with the cardiac‐specific α‐subunit SCN5a in Xenopus oocytes resulted in a threefold increase in the level of functional sodium channel expression, similar to that observed when SCN1b was co‐expressed with SCN5a. These results suggest that both SCN1b and SCN3b improve the efficiency with which the mature channel is targeted to the plasma membrane. 4 When measured in cell‐attached oocyte macropatches, SCN3b caused a significant depolarising shift in the half‐voltage of steady‐state inactivation compared to SCN5a alone or SCN5a + SCN1b. The half‐voltage of steady‐state activation was not significantly different between SCN5a alone and SCN5a + SCN3b or SCN5a + SCN1b. 5 The rates of inactivation for SCN5a co‐expressed with either subunit were not significantly different from that for SCN5a alone. However, recovery from inactivation at −90 mV was significantly faster for SCN5a + SCN1b compared to SCN5a + SCN3b, and both were significantly faster than SCN5a alone. 6 Thus, SCN1b and SCN3b have distinctive effects on the kinetics of activation and inactivation, which, in combination with the different patterns of expression of SCN3b and SCN1b, could have important consequences for the integrated electrical activity of the intact heart.

Clathrin promotes incorporation of cargo into coated pits by activation of the AP2 adaptor μ2 kinase

Endocytic cargo such as the transferrin receptor is incorporated into clathrin-coated pits by associating, via tyrosine-based motifs, with the AP2 complex. Cargo–AP2 interactions occur via the μ2 subunit of AP2, which needs to be phosphorylated for endocytosis to occur. The most likely role for μ2 phosphorylation is in cargo recruitment because μ2 phosphorylation enhances its binding to internalization motifs. Here, we investigate the control of μ2 phosphorylation. We identify clathrin as a specific activator of the μ2 kinase and, in permeabilized cells, we show that ligand sequestration, driven by exogenous clathrin, results in elevated levels of μ2 phosphorylation. Furthermore, we show that AP2 containing phospho-μ2 is mainly associated with assembled clathrin in vivo, and that the level of phospho-μ2 is strongly reduced in a chicken B cell line depleted of clathrin heavy chain. Our results imply a central role for clathrin in the regulation of cargo selection via the modulation of phospho-μ2 levels.

Trafficking and cellular distribution of voltage-gated sodium channels.

Electrical excitability in cells such as neurons and myocytes depends not only upon the expression of voltage‐gated sodium channels but also on their correct targeting within the plasma membrane. Placing sodium channels within a broader cell biological context is beginning to shed new light on a variety of important questions such as the integration of neuronal signaling. Mutations that affect sodium channel trafficking have been shown to underlie several life‐threatening conditions including cardiac arrhythmias, revealing an important clinical context to these studies.

Secretory granule biogenesis in sympathoadrenal cells : Identification of a granulogenic determinant in the secretory prohormone chromogranin A

Chromogranin A (CgA) may be critical for secretory granule biogenesis in sympathoadrenal cells. We found that silencing the expression of CgA reduced the number of secretory granules in normal sympathoadrenal cells (PC12), and we therefore questioned whether a discrete domain of CgA might promote the formation of a regulated secretory pathway in variant sympathoadrenal cells (A35C) devoid of such a phenotype. The secretory granule-forming activity of a series of human CgA domains labeled with a hemagglutinin epitope, green fluorescent protein, or embryonic alkaline phosphatase was assessed in A35C cells by deconvolution and electron microscopy and by secretagogue-stimulated release assays. Expression of CgA in A35C cells induced the formation of vesicular organelles throughout the cytoplasm, whereas two constitutive secretory pathway markers accumulated in the Golgi complex. The lysosome-associated membrane protein LGP110 did not co-localize with CgA, consistent with non-lysosomal targeting of the granin in A35C cells. Thus, CgA-expressing A35C cells showed electron-dense granules ∼180-220 nm in diameter, and secretagogue-stimulated exocytosis of CgA from A35C cells suggested that expression of the granin may be sufficient to restore a regulated secretory pathway and thereby rescue the sorting of other secretory proteins. We show that the formation of vesicular structures destined for regulated exocytosis may be mediated by a determinant located within the CgA N-terminal region (CgA-(1-115), with a necessary contribution of CgA-(40-115)), but not the C-terminal region (CgA-(233-439)) of the protein. We propose that CgA promotes the biogenesis of secretory granules by a mechanism involving a granulogenic determinant located within CgA-(40-115) of the mature protein.

Crystal structure and molecular imaging of the Nav channel β3 subunit indicates a trimeric assembly.

Background: The vertebrate sodium channel β3 subunit regulates channel behavior. Results: The immunoglobulin domain of the human β3 subunit crystallizes as a trimer, and the full-length protein assembles as a trimer in vivo. Conclusion: Our results reveal an unexpected organization of the β3 subunit. Significance: A new structural insight into the sodium channel is presented. The vertebrate sodium (Nav) channel is composed of an ion-conducting α subunit and associated β subunits. Here, we report the crystal structure of the human β3 subunit immunoglobulin (Ig) domain, a functionally important component of Nav channels in neurons and cardiomyocytes. Surprisingly, we found that the β3 subunit Ig domain assembles as a trimer in the crystal asymmetric unit. Analytical ultracentrifugation confirmed the presence of Ig domain monomers, dimers, and trimers in free solution, and atomic force microscopy imaging also detected full-length β3 subunit monomers, dimers, and trimers. Mutation of a cysteine residue critical for maintaining the trimer interface destabilized both dimers and trimers. Using fluorescence photoactivated localization microscopy, we detected full-length β3 subunit trimers on the plasma membrane of transfected HEK293 cells. We further show that β3 subunits can bind to more than one site on the Nav 1.5 α subunit and induce the formation of α subunit oligomers, including trimers. Our results suggest a new and unexpected role for the β3 subunits in Nav channel cross-linking and provide new structural insights into some pathological Nav channel mutations.

A new look at sodium channel β subunits

Voltage-gated sodium (Nav) channels are intrinsic plasma membrane proteins that initiate the action potential in electrically excitable cells. They are a major focus of research in neurobiology, structural biology, membrane biology and pharmacology. Mutations in Nav channels are implicated in a wide variety of inherited pathologies, including cardiac conduction diseases, myotonic conditions, epilepsy and chronic pain syndromes. Drugs active against Nav channels are used as local anaesthetics, anti-arrhythmics, analgesics and anti-convulsants. The Nav channels are composed of a pore-forming α subunit and associated β subunits. The β subunits are members of the immunoglobulin (Ig) domain family of cell-adhesion molecules. They modulate multiple aspects of Nav channel behaviour and play critical roles in controlling neuronal excitability. The recently published atomic resolution structures of the human β3 and β4 subunit Ig domains open a new chapter in the study of these molecules. In particular, the discovery that β3 subunits form trimers suggests that Nav channel oligomerization may contribute to the functional properties of some β subunits.

The Organelle Proteome of the DT40 Lymphocyte Cell Line

A major challenge in eukaryotic cell biology is to understand the roles of individual proteins and the subcellular compartments in which they reside. Here, we use the localization of organelle proteins by isotope tagging technique to complete the first proteomic analysis of the major organelles of the DT40 lymphocyte cell line. This cell line is emerging as an important research tool because of the ease with which gene knockouts can be generated. We identify 1090 proteins through the analysis of preparations enriched for integral membrane or soluble and peripherally associated proteins and localize 223 proteins to the endoplasmic reticulum, Golgi, lysosome, mitochondrion, or plasma membrane by matching their density gradient distributions to those of known organelle residents. A striking finding is that within the secretory and endocytic pathway a high proportion of proteins are not uniquely localized to a single organelle, emphasizing the dynamic steady-state nature of intracellular compartments in eukaryotic cells.

Scn3b knockout mice exhibit abnormal sino‐atrial and cardiac conduction properties

Aim:  In contrast to extensive reports on the roles of Nav1.5 α‐subunits, there have been few studies associating the β‐subunits with cardiac arrhythmogenesis. We investigated the sino‐atrial and conduction properties in the hearts of Scn3b−/− mice.

A role for the transcriptional repressor REST in maintaining the phenotype of neurosecretory‐deficient PC12 cells

The rat PC12 variant cell line, A35C, lacks regulated secretory organelles due to a selective transcriptional block. Hence, A35C may provide clues about the mechanisms that underlie control of neurosecretion. We used mRNA microarray profiling to examine gene expression in A35C. Genes for regulated secretory proteins were down‐regulated, while other membrane trafficking pathways were unaffected. A subset of genes repressed in A35C contain binding sites for the neuronal transcriptional repressor, RE1‐silencing transcription factor (REST), and REST is expressed in A35C but not normal PC12 cells. Blocking the activity of REST in A35C using a dominant‐negative construct induced the reappearance of mRNAs for synaptophysin, chromogranin A, synaptotagmin IV and the β3 subunit of the voltage‐gated sodium channel (Scn3b), all of which contain RE1 sites in their genes. In the case of Scn3b, the corresponding protein was also re‐expressed. Granule and synaptic vesicle proteins were not re‐expressed at the protein level, despite reactivation of their mRNA, suggesting the existence of additional post‐transcriptional control for these proteins. Our work identifies one of the mechanisms underlying the phenotype of neurosecretory‐deficient neuroendocrine cells, and begins to define the critical components that determine a key aspect of the neuroendocrine phenotype.

New Insights into the DT40 B Cell Receptor Cluster Using a Proteomic Proximity Labeling Assay

Background: B cell receptor (BCR) clusters modulate BCR signaling in B-lymphocytes. Results: We used a quantitative proteomic proximity assay to analyze the BCR cluster in DT40 cells. Conclusion: Our proximity labeling assay identified novel components of the BCR cluster linked to integrin signaling. Significance: We provide new insights into BCR assembly and identify new and unexpected targets for further functional analysis. In the vertebrate immune system, each B-lymphocyte expresses a surface IgM-class B cell receptor (BCR). When cross-linked by antigen or anti-IgM antibody, the BCR accumulates with other proteins into distinct surface clusters that activate cell signaling, division, or apoptosis. However, the molecular composition of these clusters is not well defined. Here we describe a quantitative assay we call selective proteomic proximity labeling using tyramide (SPPLAT). It allows proteins in the immediate vicinity of a target to be selectively biotinylated, and hence isolated for mass spectrometry analysis. Using the chicken B cell line DT40 as a model, we use SPPLAT to provide the first proteomic analysis of any BCR cluster using proximity labeling. We detect known components of the BCR cluster, including integrins, together with proteins not previously thought to be BCR-associated. In particular, we identify the chicken B-lymphocyte allotypic marker chB6. We show that chB6 moves to within about 30–40 nm of the BCR following BCR cross-linking, and we show that cross-linking chB6 activates cell binding to integrin substrates laminin and gelatin. Our work provides new insights into the nature and composition of the BCR cluster, and confirms SPPLAT as a useful research tool in molecular and cellular proteomics.