Elizabeth M. Fitzgerald

cAMP-dependent phosphorylation of the tetrodotoxin-resistant voltage-dependent sodium channel, SNS

1 Protein kinase A (PKA) modulation of tetrodotoxin‐resistant (TTX‐r) voltage‐gated sodium channels may underly the hyperalgesic responses of mammalian sensory neurones. We have therefore examined PKA phosphorylation of the cloned α‐subunit of the rat sensory neurone‐specific TTX‐r channel SNS. Phosphorylation of SNS was compared with that of a mutant channel, SNS(SA), in which all five PKA consensus sites (RXXS) within the intracellular I‐II loop had been eliminated by site‐directed mutagenesis (serine to alanine). 2 In vitro PKA phosphorylation and tryptic peptide mapping of SNS and mutant SNS(SA) I‐II loops expressed as glutathione‐S‐transferase (GST) fusion proteins confirmed that the five mutated serines were the major PKA substrates within the SNS I‐II loop. 3 SNS and SNS(SA) channels were transiently expressed in COS‐7 cells and their electrophysiological properties compared. In wild‐type SNS channels, forskolin and 8‐bromo cAMP produced effects consistent with PKA phosphorylation. Mutant SNS(SA) currents, however, were not significantly affected by either agent. Thus, elimination of the I‐II loop PKA consensus sites caused a marked reduction in PKA modulation of wild‐type channels. 4 Under control conditions, the voltage dependence of activation of SNS(SA) current was shifted to depolarized potentials compared with SNS. This was associated with a slowing of SNS(SA) current inactivation at hyperpolarized potentials and suggested a tonic PKA phosphorylation of wild‐type channels under basal conditions. 5 We conclude that the major substrates involved in functional PKA modulation of the SNS channel are located within the intracellular I‐II loop.

Regulation of voltage-gated sodium channel expression in cancer: hormones, growth factors and auto-regulation.

Although ion channels are increasingly being discovered in cancer cells in vitro and in vivo, and shown to contribute to different aspects and stages of the cancer process, much less is known about the mechanisms controlling their expression. Here, we focus on voltage-gated Na+ channels (VGSCs) which are upregulated in many types of carcinomas where their activity potentiates cell behaviours integral to the metastatic cascade. Regulation of VGSCs occurs at a hierarchy of levels from transcription to post-translation. Importantly, mainstream cancer mechanisms, especially hormones and growth factors, play a significant role in the regulation. On the whole, in major hormone-sensitive cancers, such as breast and prostate cancer, there is a negative association between genomic steroid hormone sensitivity and functional VGSC expression. Activity-dependent regulation by positive feedback has been demonstrated in strongly metastatic cells whereby the VGSC is self-sustaining, with its activity promoting further functional channel expression. Such auto-regulation is unlike normal cells in which activity-dependent regulation occurs mostly via negative feedback. Throughout, we highlight the possible clinical implications of functional VGSC expression and regulation in cancer.

Regulation of voltage-dependent calcium channels in rat sensory neurones involves a Ras–mitogen-activated protein kinase pathway

1 The small G‐protein Ras, a critical component in the signalling pathways regulating cell growth, is involved in the tonic upregulation of voltage‐dependent calcium channels (VDCCs) in rat sensory neurones. To investigate which downstream effector(s) of Ras is involved in this process, a series of Ras mutant cDNAs were co‐expressed with green fluorescent protein (GFP) in primary cultured rat dorsal root ganglion neurones (DRGs). 2 Constitutively active V12Ras (glycine 12 to valine) markedly increased basal calcium current density by 41 % compared with control cells (GFP alone). In contrast, a farnesylation‐defective mutant, V12S186Ras (cysteine 186 to serine; activates no downstream effectors), significantly reduced calcium current density by 47 %. 3 Ras effector region mutants V12C40 (tyrosine 40 to cysteine; activates the p110 α‐subunit of phosphatidylinositol 3‐kinase) and V12G37 (glutamic acid 37 to glycine; activates Ral guanine nucleotide dissociation stimulator) had no significant effect on VDCC current. However, V12S35Ras (threonine 35 to serine; activates Raf‐1 and the mitogen‐activated protein kinase (MAPK) pathway) markedly increased basal calcium current density by 67 %, suggesting that Raf‐1 activation is sufficient for Ras enhancement of calcium current in these cells. 4 Raf‐1 activates MEK (MAPK kinase) in the MAPK pathway, and the MEK inhibitor U0126 reduced calcium current by 45 % after 10–15 min, whereas the inactive analogue U0124 had no effect. This rapid time course for MEK inhibition suggests direct modulation of VDCCs via the Ras‐MAPK pathway rather than gene expression‐mediated effects. 5 The relative proportions of ω‐conotoxin GVIA‐ and nicardipine‐sensitive N‐ (∼40 %) and L‐ (∼40 %) type currents were unaffected by either V12S35Ras expression or U0126 pre‐treatment, suggesting that all components of calcium current in DRGs, are enhanced via this pathway.

Functional expression of the voltage-gated Na+-channel Nav1.7 is necessary for EGF-mediated invasion in human non-small cell lung cancer cells

Summary Various ion channels are expressed in human cancers where they are intimately involved in proliferation, angiogenesis, invasion and metastasis. Expression of functional voltage-gated Na+ channels (Nav) is implicated in the metastatic potential of breast, prostate, lung and colon cancer cells. However, the cellular mechanisms that regulate Nav expression in cancer remain largely unknown. Growth factors are attractive candidates; they not only play crucial roles in cancer progression but are also key regulators of ion channel expression and activity in non-cancerous cells. Here, we examine the role of epidermal growth factor receptor (EGFR) signalling and Nav in non-small cell lung carcinoma (NSCLC) cell lines. We show unequivocally, that functional expression of the &agr; subunit Nav1.7 promotes invasion in H460 NSCLC cells. Inhibition of Nav1.7 activity (using tetrodotoxin) or expression (by using small interfering RNA), reduces H460 cell invasion by up to 50%. Crucially, non-invasive wild type A549 cells lack functional Nav, whereas exogenous overexpression of the Nav1.7 &agr; subunit is sufficient to promote TTX-sensitive invasion of these cells. EGF/EGFR signalling enhances proliferation, migration and invasion of H460 cells but we find that, specifically, EGFR-mediated upregulation of Nav1.7 is necessary for invasive behaviour in these cells. Examination of Nav1.7 expression at mRNA, protein and functional levels further reveals that EGF/EGFR signalling via the ERK1/2 pathway controls transcriptional regulation of channel expression to promote cellular invasion. Immunohistochemistry of patient biopsies confirms the clinical relevance of Nav1.7 expression in NSCLC. Thus, Nav1.7 has significant potential as a new target for therapeutic intervention and/or as a diagnostic or prognostic marker in NSCLC.

Targeting of Voltage-Gated Calcium Channel α2δ-1 Subunit to Lipid Rafts Is Independent from a GPI-Anchoring Motif

Voltage-gated calcium channels (Cav) exist as heteromultimers comprising a pore-forming α1 with accessory β and α2δ subunits which modify channel trafficking and function. We previously showed that α2δ-1 (and likely the other mammalian α2δ isoforms - α2δ-2, 3 and 4) is required for targeting Cavs to lipid rafts, although the mechanism remains unclear. Whilst originally understood to have a classical type I transmembrane (TM) topology, recent evidence suggests the α2δ subunit contains a glycosylphosphatidylinositol (GPI)-anchor that mediates its association with lipid rafts. To test this notion, we have used a strategy based on the expression of chimera, where the reported GPI-anchoring sequences in the gabapentinoid-sensitive α2δ-1 subunit have been substituted with those of a functionally inert Type I TM-spanning protein – PIN-G. Using imaging, electrophysiology and biochemistry, we find that lipid raft association of PIN-α2δ is unaffected by substitution of the GPI motif with the TM domain of PIN-G. Moreover, the presence of the GPI motif alone is not sufficient for raft localisation, suggesting that upstream residues are required. GPI-anchoring is susceptible to phosphatidylinositol-phospholipase C (PI-PLC) cleavage. However, whilst raft localisation of PIN-α2δ is disrupted by PI-PLC treatment, this is assay-dependent and non-specific effects of PI-PLC are observed on the distribution of the endogenous raft marker, caveolin, but not flotillin. Taken together, these data are most consistent with a model where α2δ-1 retains its type I transmembrane topology and its targeting to lipid rafts is governed by sequences upstream of the putative GPI anchor, that promote protein-protein, rather than lipid-lipid interactions.

The presence of ca2+ channel β subunit is required for mitogen‐activated protein kinase (mapk)‐dependent modulation of α1b ca2+ channels in cos‐7 cells

In rat sensory neurones, voltage‐dependent calcium channels (VDCCs), including the N‐type, are tonically up‐regulated via Ras/mitogen‐activated protein kinase (MAPK) signalling. To determine whether VDCC β subunit is involved in this process, the role of the four neuronal βs (β1b, β2a, β3, β4) in MAPK‐dependent modulation of α1B (Cav2.2, N‐type) Ca2+ channels has been examined in COS‐7 cells. MAPK is exclusively activated by MAPK kinase (MEK), and here, acute application of a MEK‐specific inhibitor UO126, significantly inhibited peak α1B Ca2+ channel current (Imax) within a period of 5–10 min, regardless of which β subunit was co‐expressed (25‐50%, P < 0.01). With β2a however, the percentage inhibition of Imax was less than that observed with any other β (ANOVA: F3,34= 6.48, P < 0.01). UO126 also caused a hyperpolarising shift (6 ± 1 mV, P < 0.001) in the voltage dependence of β2a current activation, such that inhibition occurred only at depolarised potentials (> +5 mV) whereas at more negative potentials the current amplitude was enhanced. A marked change in β2a current kinetics, perceived either as decreased activation or increased inactivation, was also associated with UO126 application. A similar effect of UO126 on β4 current kinetics was also observed. The β2a‐specific effects of UO126 on current inhibition and voltage dependence of activation were abolished when α1B was co‐expressed with de‐palmitoylated β2a(C3,4S), in which amino terminal cysteines 3 and 4 had been mutated to serines. In the absence of β subunit, UO126 had no effect on α1B Ca2+ channel current. Together, these data suggest an absolute requirement for β in MAPK‐dependent modulation of these channels. Since β subunits vary both in their temporal expression and localisation within neurones, β subunit‐dependent modulation of N‐type Ca2+ channels via MAPK could provide an important new mechanism by which to fine‐tune neurotransmitter release.

The R-Domain: Identification of an N-terminal Region of the α 2 δ-1 Subunit Which is Necessary and Sufficient for its Effects on Ca v 2.2 Calcium Currents

Voltage-gated calcium channels (Cav) and their associated proteins are pivotal signalling complexes in excitable cell physiology. In nerves and muscle, Cav tailor calcium influx to processes including neurotransmission, muscle contraction and gene expression. Cav comprise a pore-forming α1 and modulatory β and α2δ subunits - the latter targeted by anti-epileptic and anti-nociceptive gabapentinoid drugs. However, the mechanisms of gabapentinoid action are unclear, not least because detailed structure-function mapping of the α2δ subunit remains lacking. Using molecular biology and electrophysiological approaches we have conducted the first systematic mapping of α2δ subunit structure-function. We generated a series of cDNA constructs encoding chimera, from which successive amino acids from the rat α2δ-1 subunit were incorporated into a Type 1 reporter protein - PIN-G, to produce sequential extensions from the transmembrane (TM) region towards the N-terminus. By successive insertion of a TGA stop codon, a further series of N- to C-terminal extension constructs lacking the TM region, were also generated. Using this approach we have defined the minimal region of α2δ-1 - we term the R-domain (Rd), that appears to contain all the machinery necessary to support the electrophysiological and trafficking effects of α2δ-1 on Cav. Structural algorithms predict that Rd is conserved across all four α2δ subunits, including RNA splice variants, and irrespective of phyla and taxa. We suggest, therefore, that Rd likely constitutes the major locus for physical interaction with the α1 subunit and may provide a target for novel Cav therapeutics.