William J. Brackenbury

Functional analysis of AtHKT1 in Arabidopsis shows that Na+ recirculation by the phloem is crucial for salt tolerance

Two allelic recessive mutations of Arabidopsis, sas2‐1 and sas2‐2, were identified as inducing sodium overaccumulation in shoots. The sas2 locus was found (by positional cloning) to correspond to the AtHKT1 gene. Expression in Xenopus oocytes revealed that the sas2‐1 mutation did not affect the ionic selectivity of the transporter but strongly reduced the macro scopic (whole oocyte current) transport activity. In Arabidopsis, expression of AtHKT1 was shown to be restricted to the phloem tissues in all organs. The sas2‐1 mutation strongly decreased Na+ concentration in the phloem sap. It led to Na+ overaccumulation in every aerial organ (except the stem), but to Na+ underaccumulation in roots. The sas2 plants displayed increased sensitivity to NaCl, with reduced growth and even death under moderate salinity. The whole set of data indicates that AtHKT1 is involved in Na+ recirculation from shoots to roots, probably by mediating Na+ loading into the phloem sap in shoots and unloading in roots, this recirculation removing large amounts of Na+ from the shoot and playing a crucial role in plant tolerance to salt.

Voltage-Gated Sodium Channel Expression and Potentiation of Human Breast Cancer Metastasis

Purpose: Ion channel activity is involved in several basic cellular behaviors that are integral to metastasis (e.g., proliferation, motility, secretion, and invasion), although their contribution to cancer progression has largely been ignored. The purpose of this study was to investigate voltage-gated Na+ channel (VGSC) expression and its possible role in human breast cancer. Experimental Design: Functional VGSC expression was investigated in human breast cancer cell lines by patch clamp recording. The contribution of VGSC activity to directional motility, endocytosis, and invasion was evaluated by in vitro assays. Subsequent identification of the VGSC α-subunit(s) expressed in vitro was achieved using reverse transcription-PCR, immunocytochemistry, and Western blot techniques and used to investigate VGSCα expression and its association with metastasis in vivo. Results: VGSC expression was significantly up-regulated in metastatic human breast cancer cells and tissues, and VGSC activity potentiated cellular directional motility, endocytosis, and invasion. Reverse transcription-PCR revealed that Nav1.5, in its newly identified “neonatal” splice form, was specifically associated with strong metastatic potential in vitro and breast cancer progression in vivo. An antibody specific for this form confirmed up-regulation of neonatal Nav1.5 protein in breast cancer cells and tissues. Furthermore, a strong correlation was found between neonatal Nav1.5 expression and clinically assessed lymph node metastasis. Conclusions: Up-regulation of neonatal Nav1.5 occurs as an integral part of the metastatic process in human breast cancer and could serve both as a novel marker of the metastatic phenotype and a therapeutic target.

Na+ Channel β Subunits: Overachievers of the Ion Channel Family

Voltage-gated Na+ channels (VGSCs) in mammals contain a pore-forming α subunit and one or more β subunits. There are five mammalian β subunits in total: β1, β1B, β2, β3, and β4, encoded by four genes: SCN1B–SCN4B. With the exception of the SCN1B splice variant, β1B, the β subunits are type I topology transmembrane proteins. In contrast, β1B lacks a transmembrane domain and is a secreted protein. A growing body of work shows that VGSC β subunits are multifunctional. While they do not form the ion channel pore, β subunits alter gating, voltage-dependence, and kinetics of VGSCα subunits and thus regulate cellular excitability in vivo. In addition to their roles in channel modulation, β subunits are members of the immunoglobulin superfamily of cell adhesion molecules and regulate cell adhesion and migration. β subunits are also substrates for sequential proteolytic cleavage by secretases. An example of the multifunctional nature of β subunits is β1, encoded by SCN1B, that plays a critical role in neuronal migration and pathfinding during brain development, and whose function is dependent on Na+ current and γ-secretase activity. Functional deletion of SCN1B results in Dravet Syndrome, a severe and intractable pediatric epileptic encephalopathy. β subunits are emerging as key players in a wide variety of physiopathologies, including epilepsy, cardiac arrhythmia, multiple sclerosis, Huntington’s disease, neuropsychiatric disorders, neuropathic and inflammatory pain, and cancer. β subunits mediate multiple signaling pathways on different timescales, regulating electrical excitability, adhesion, migration, pathfinding, and transcription. Importantly, some β subunit functions may operate independently of α subunits. Thus, β subunits perform critical roles during development and disease. As such, they may prove useful in disease diagnosis and therapy.

Membrane potential and cancer progression

Membrane potential (Vm), the voltage across the plasma membrane, arises because of the presence of different ion channels/transporters with specific ion selectivity and permeability. Vm is a key biophysical signal in non-excitable cells, modulating important cellular activities, such as proliferation and differentiation. Therefore, the multiplicities of various ion channels/transporters expressed on different cells are finely tuned in order to regulate the Vm. It is well-established that cancer cells possess distinct bioelectrical properties. Notably, electrophysiological analyses in many cancer cell types have revealed a depolarized Vm that favors cell proliferation. Ion channels/transporters control cell volume and migration, and emerging data also suggest that the level of Vm has functional roles in cancer cell migration. In addition, hyperpolarization is necessary for stem cell differentiation. For example, both osteogenesis and adipogenesis are hindered in human mesenchymal stem cells (hMSCs) under depolarizing conditions. Therefore, in the context of cancer, membrane depolarization might be important for the emergence and maintenance of cancer stem cells (CSCs), giving rise to sustained tumor growth. This review aims to provide a broad understanding of the Vm as a bioelectrical signal in cancer cells by examining several key types of ion channels that contribute to its regulation. The mechanisms by which Vm regulates cancer cell proliferation, migration, and differentiation will be discussed. In the long term, Vm might be a valuable clinical marker for tumor detection with prognostic value, and could even be artificially modified in order to inhibit tumor growth and metastasis.

An Emerging Role for Voltage-Gated Na+ Channels in Cellular Migration: Regulation of Central Nervous System Development and Potentiation of Invasive Cancers

Voltage-gated Na+ channels (VGSCs) exist as macromolecular complexes containing a pore-forming α subunit and one or more β subunits. The VGSC α subunit gene family consists of 10 members, which have distinct tissue-specific and developmental expression profiles. So far, four β subunits (β1—β4) and one splice variant of β1 (β1A, also called β1B) have been identified. VGSC β subunits are multifunctional, serving as modulators of channel activity, regulators of channel cell surface expression, and as members of the immunoglobulin superfamily, cell adhesion molecules (CAMs). β subunits are substrates of β-amyloid precursor protein-cleaving enzyme (BACE1) and γ-secretase, yielding intracellular domains (ICDs) that may further modulate cellular activity via transcription. Recent evidence shows that β1 regulates migration and pathfinding in the developing postnatal CNS in vivo. The α and β subunits, together with other components of the VGSC signaling complex, may have dynamic interactive roles depending on cell/tissue type, developmental stage, and pathophysiology. In addition to excitable cells like nerve and muscle, VGSC α and β subunits are functionally expressed in cells that are traditionally considered nonexcitable, including glia, vascular endothelial cells, and cancer cells. In particular, the α subunits are up-regulated in line with metastatic potential and are proposed to enhance cellular migration and invasion. In contrast to the α subunits, β1 is more highly expressed in weakly metastatic cancer cells, and evidence suggests that its expression enhances cellular adhesion. Thus, novel roles are emerging for VGSC α and β subunits in regulating migration during normal postnatal development of the CNS as well as during cancer metastasis.

Functional reciprocity between Na+ channel Nav1.6 and β1 subunits in the coordinated regulation of excitability and neurite outgrowth

Voltage-gated Na+ channel (VGSC) β1 subunits regulate cell–cell adhesion and channel activity in vitro. We previously showed that β1 promotes neurite outgrowth in cerebellar granule neurons (CGNs) via homophilic cell adhesion, fyn kinase, and contactin. Here we demonstrate that β1-mediated neurite outgrowth requires Na+ current (INa) mediated by Nav1.6. In addition, β1 is required for high-frequency action potential firing. Transient INa is unchanged in Scn1b (β1) null CGNs; however, the resurgent INa, thought to underlie high-frequency firing in Nav1.6-expressing cerebellar neurons, is reduced. The proportion of axon initial segments (AIS) expressing Nav1.6 is reduced in Scn1b null cerebellar neurons. In place of Nav1.6 at the AIS, we observed an increase in Nav1.1, whereas Nav1.2 was unchanged. This indicates that β1 is required for normal localization of Nav1.6 at the AIS during the postnatal developmental switch to Nav1.6-mediated high-frequency firing. In agreement with this, β1 is normally expressed with α subunits at the AIS of P14 CGNs. We propose reciprocity of function between β1 and Nav1.6 such that β1-mediated neurite outgrowth requires Nav1.6-mediated INa, and Nav1.6 localization and consequent high-frequency firing require β1. We conclude that VGSC subunits function in macromolecular signaling complexes regulating both neuronal excitability and migration during cerebellar development.

Voltage-Gated Na+ Channel β1 Subunit-Mediated Neurite Outgrowth Requires Fyn Kinase and Contributes to Postnatal CNS Development In Vivo

Voltage-gated Na+ channel β1 subunits are multifunctional, participating in channel modulation and cell adhesion in vitro. We previously demonstrated that β1 promotes neurite outgrowth of cultured cerebellar granule neurons (CGNs) via homophilic adhesion. Both lipid raft-associated kinases and nonraft fibroblast growth factor (FGF) receptors are implicated in cell adhesion molecule-mediated neurite extension. In the present study, we reveal that β1-mediated neurite outgrowth is abrogated in Fyn and contactin (Cntn) null CGNs. β1 protein levels are unchanged in Fyn null brains, whereas levels are significantly reduced in Cntn null brain lysates. FGF or EGF (epidermal growth factor) receptor kinase inhibitors have no effect on β1-mediated neurite extension. These results suggest that β1-mediated neurite outgrowth occurs through a lipid raft signaling mechanism that requires the presence of both fyn kinase and contactin. In vivo, Scn1b null mice show defective CGN axon extension and fasciculation indicating that β1 plays a role in cerebellar microorganization. In addition, we find that axonal pathfinding and fasciculation are abnormal in corticospinal tracts of Scn1b null mice consistent with the suggestion that β1 may have widespread effects on postnatal neuronal development. These data are the first to demonstrate a cell-adhesive role for β1 in vivo. We conclude that voltage-gated Na+ channel β1 subunits signal via multiple pathways on multiple timescales and play important roles in the postnatal development of the CNS.

Voltage-gated sodium channels and metastatic disease.

Voltage-gated Na+ channels (VGSCs) are macromolecular protein complexes containing a pore-forming α subunit and smaller non-pore-forming β subunits. VGSCs are expressed in metastatic cells from a number of cancers. In these cells, Na+ current carried by α subunits enhances migration, invasion and metastasis in vivo. In contrast, the β subunits mediate cellular adhesion and process extension. The prevailing hypothesis is that VGSCs are upregulated in cancer, in general favoring an invasive/metastatic phenotype, although the mechanisms are still not fully clear. Expression of the Nav1.5 α subunit associates with poor prognosis in clinical breast cancer specimens, suggesting that VGSCs may have utility as prognostic markers for cancer progression. Furthermore, repurposing existing VGSC-blocking therapeutic drugs may provide a new strategy to improve outcomes in patients suffering from metastatic disease, which is the major cause of cancer-related deaths, and for which there is currently no cure.

Voltage-gated Na+ channels: Potential for β subunits as therapeutic targets

Background: Voltage gated Na+ channels (VGSCs) contain a pore-forming α subunit and one or more β subunits. VGSCs are involved in a wide variety of pathophysiologies, including epilepsy, cardiac arrhythmia, multiple sclerosis, periodic paralysis, migraine, neuropathic and inflammatory pain, Huntingtons disease and cancer. Increasing evidence implicates the β subunits as key players in these disorders. Objective: To review the recent literature describing the multifunctional roles of VGSC β subunits in the context of their role(s) in disease. Methods: An extensive review of the literature on β subunits. Results/conclusion: β subunits are multifunctional. As components of VGSC complexes, β subunits mediate signaling processes regulating electrical excitability, adhesion, migration, pathfinding and transcription. β subunits may prove useful in disease diagnosis and therapy.

Voltage-Gated Na+ Channel β1B: A Secreted Cell Adhesion Molecule Involved in Human Epilepsy

Scn1b-null mice have a severe neurological and cardiac phenotype. Human mutations in SCN1B result in epilepsy and cardiac arrhythmia. SCN1B is expressed as two developmentally regulated splice variants, β1 and β1B, that are each expressed in brain and heart in rodents and humans. Here, we studied the structure and function of β1B and investigated a novel human SCN1B epilepsy-related mutation (p.G257R) unique to β1B. We show that wild-type β1B is not a transmembrane protein, but a soluble protein expressed predominantly during embryonic development that promotes neurite outgrowth. Association of β1B with voltage-gated Na+ channels Nav1.1 or Nav1.3 is not detectable by immunoprecipitation and β1B does not affect Nav1.3 cell surface expression as measured by [3H]saxitoxin binding. However, β1B coexpression results in subtle alteration of Nav1.3 currents in transfected cells, suggesting that β1B may modulate Na+ current in brain. Similar to the previously characterized p.R125C mutation, p.G257R results in intracellular retention of β1B, generating a functional null allele. In contrast, two other SCN1B mutations associated with epilepsy, p.C121W and p.R85H, are expressed at the cell surface. We propose that β1B p.G257R may contribute to epilepsy through a mechanism that includes intracellular retention resulting in aberrant neuronal pathfinding.