Neonatal NaV 1.5 channels: pharmacological distinctiveness of a cancer-related voltage-gated sodium channel splice variant.

BACKGROUND AND PURPOSE: Voltage-gated sodium (NaV ) channels are expressed de novo in carcinomas where their activity promotes invasiveness. Breast and colon cancer cells express the neonatal splice variant of NaV 1.5 (nNaV 1.5), which has several amino acid substitutions in the domain I voltage-sensor compared with its adult counterpart (aNaV 1.5). This study aimed to determine whether nNaV 1.5 channels could be distinguished pharmacologically from aNaV 1.5 channels. EXPERIMENTAL APPROACH: Cells expressing either nNaV 1.5 or aNaV 1.5 channels were exposed to low MW inhibitors, an antibody or natural toxins, and changes in electrophysiological parameters were measured. Stable expression in EBNA cells and transient expression in Xenopus laevis oocytes were used. Currents were recorded by whole-cell patch clamp and two-electrode voltage-clamp, respectively. KEY RESULTS: Several clinically used blockers of NaV channels (lidocaine, procaine, phenytoin, mexiletine, ranolazine, and riluzole) could not distinguish between nNaV 1.5 or aNaV 1.5 channels. However, two tarantula toxins (HaTx and ProTx-II) and a polyclonal antibody (NESOpAb) preferentially inhibited currents elicited by either nNaV 1.5 or aNaV 1.5 channels by binding to the spliced region of the channel. Furthermore, the amino acid residue at position 211 (aspartate in aNaV 1.5/lysine in nNaV 1.5), that is, the charge reversal in the spliced region of the channel, played a key role in the selectivity, especially in antibody binding. CONCLUSION AND IMPLICATIONS: We conclude that the cancer-related nNaV 1.5 channel can be distinguished pharmacologically from its nearest neighbour, aNaV 1.5 channels. Thus, it may be possible to design low MW compounds as antimetastatic drugs for non-toxic therapy of nNaV 1.5-expressing carcinomas.

Cationic Modulation of Voltage-Gated Sodium Channel (Nav1.5): Neonatal Versus Adult Splice Variants-1. Monovalent (H+) Ions.

Background: Voltage-gated sodium channels are functionally expressed in human carcinomas. In breast and colon cancers, the neonatal splice variant of Nav1.5 (nNav1.5) is dominant. This differs from the adult (aNav1.5) by several amino acids, including an outer charge reversal (residue-211): negatively charged aspartate (aNav1.5) versus positively charged lysine (nNav1.5). Thus, nNav1.5 and aNav1.5 may respond to extracellular charges differently. Materials and Methods: We used whole-cell patch-clamp recording to compare the electrophysiological effects of the monovalent cation hydrogen (H+) on nNav1.5 and aNav1.5 expressed stably in EBNA cells. Results: Increasing the H+ concentration (acidifying pH) reduced channel conductance and inhibited peak currents. Also, there was a positive shift in the voltage dependence of activation. These changes were significantly smaller for nNav1.5, compared with aNav1.5. Conclusions: nNav1.5 was more resistant to the suppressive effects of acidification compared with aNav1.5. Thus, nNav1.5 may have an advantage in promoting metastasis from the acidified tumor microenvironment.

Cationic Modulation of Voltage-Gated Sodium Channel (Nav1.5): Neonatal Versus Adult Splice Variants-2. Divalent (Cd2+) and Trivalent (Gd3+) Ions.

Background: A "neonatal" splice-form of the voltage-gated sodium channel Nav1.5 is functionally expressed in human cancers and potentiates metastatic cell behaviors. Splicing causes the replacement of 7 amino acids, including a negatively charged aspartate211 in the "adult" Nav1.5 (aNav1.5) to a positively charged lysine in the "neonatal" (nNav1.5). These changes occur in the region surrounding the DI:S3-S4 extracellular linker. The splice variants respond differently to changes in extracellular H+ and this could be of pathophysiological significance. However, how the two differentially charged splice variants would react to cations of higher valency is not known. Materials and Methods: We used patch-clamp recording to compare the electrophysiological effects of Cd2+ and Gd3+ on "adult" and "neonatal" Nav1.5 expressed stably in EBNA-293 cells. Several parameters were determined for the two channels and statistically compared. Results: Both cations inhibited peak I Na through reducing G max and induced a positive shift in the voltage range of activation. However, unlike Gd3+, Cd2+ had only a weak effect on voltage dependence of activation, and no effect on voltage dependence of inactivation, recovery from inactivation, or the kinetics of activation/inactivation. Conclusions: The electrophysiological effects of Cd2+ and Gd3+ studied were essentially the same for "neonatal" and "adult" Nav1.5, although these splice variants possess differences in their external charges. In contrast, the effects of H+ were shown earlier to be significantly differential. Taken together, these results suggest that limited adjustment of the charged structure of pharmacological agents could enable selective targeting of neonatal Nav1.5 associated with several cancers.

Intracellular calcium oscillations in strongly metastatic human breast and prostate cancer cells: control by voltage-gated sodium channel activity.

The possible association of intracellular Ca2+ with metastasis in human cancer cells is poorly understood. We have studied Ca2+ signaling in human prostate and breast cancer cell lines of strongly versus weakly metastatic potential in a comparative approach. Intracellular free Ca2+ was measured using a membrane-permeant fluorescent Ca2+-indicator dye (Fluo-4 AM) and confocal microscopy. Spontaneous Ca2+ oscillations were observed in a proportion of strongly metastatic human prostate and breast cancer cells (PC-3M and MDA-MB-231, respectively). In contrast, no such oscillations were observed in weakly/non metastatic LNCaP and MCF-7 cells, although a rise in the resting Ca2+ level could be induced by applying a high-K+ solution. Various parameters of the oscillations depended on extracellular Ca2+ and voltage-gated Na+ channel activity. Treatment with either tetrodotoxin (a general blocker of voltage-gated Na+ channels) or ranolazine (a blocker of the persistent component of the channel current) suppressed the Ca2+ oscillations. It is concluded that the functional voltage-gated Na+ channel expression in strongly metastatic cancer cells makes a significant contribution to generation of oscillatory intracellular Ca2+ activity. Possible mechanisms and consequences of the Ca2+ oscillations are discussed.

Persistent current blockers of voltage-gated sodium channels: a clinical opportunity for controlling metastatic disease.

A range of experimental and clinical data suggests strongly (i) that metastatic progression in carcinomas is accompanied (maybe even preceded) by upregulation of functional voltage-gated sodium channels (VGSCs) and (ii) that VGSC activity enhances cancer cell invasiveness. First, this review outlines the available in vitro and in vivo evidence for the VGSC expression and its proposed pathophysiological role. Second, we question the mechanism(s) whereby VGSC activity can induce such a cancer-promoting effect. We advance the hypothesis that it is the hypoxia-sensitive persistent component of the VGSC current (INaP) that is central to the phenomenon. Indeed, blockers of INaP are very effective in suppressing cancer cell invasiveness in vitro. Based upon these data, UK and international patent applications have been filed which describe the use of INaP blockers, like ranolazine ("Ranexa") and riluzole ("Rilutex"), as anti-metastatic agents. Importantly, since these drugs are already in clinical use, against conditions like cardiac angina and amyotrophic lateral scelerosis, there are no issues of dosage, unacceptable side effects or long-term use. Thus, INaP blockers have the potential to turn cancer into a chronic condition.

Oxidative stress in the in vivo DMBA rat model of breast cancer: suppression by a voltage-gated sodium channel inhibitor (RS100642).

Breast cancer (BCa) was induced in vivo in female rats with 7,12-dimethylbenz(a)anthracene (DMBA). Two main questions were addressed. Firstly, would the carcinogenesis be accompanied by oxidative stress as signalled by superoxide dismutase, glutathione peroxidase, malondialdehyde and total nitrate? Secondly, would treating the rats additionally with a blocker of voltage-gated sodium channel (VGSC) activity, shown previously to promote BCa progression, affect the oxidative responses? The DMBA-induced increases in the antioxidant systems were completely blocked by the VGSC inhibitor RS100642, which also significantly prolonged the lifespan. We conclude that VGSC inhibition in vivo can significantly protect against oxidative stress and improve survival from tumour burden.

Estrogen and non-genomic upregulation of voltage-gated Na(+) channel activity in MDA-MB-231 human breast cancer cells: role in adhesion.

External (but not internal) application of beta-estradiol (E2) increased the current amplitude of voltage-gated Na(+) channels (VGSCs) in MDA-MB-231 human breast cancer (BCa) cells. The G-protein activator GTP-gamma-S, by itself, also increased the VGSC current whilst the G-protein inhibitor GDP-beta-S decreased the effect of E2. Expression of GPR30 (a G-protein-coupled estrogen receptor) in MDA-MB-231 cells was confirmed by PCR, Western blot and immunocytochemistry. Importantly, G-1, a specific agonist for GPR30, also increased the VGSC current amplitude in a dose-dependent manner. Transfection and siRNA-silencing of GPR30 expression resulted in corresponding changes in GPR30 protein expression but only internally, and the response to E2 was not affected. The protein kinase A inhibitor, PKI, abolished the effect of E2, whilst forskolin, an adenylate cyclase activator, by itself, increased VGSC activity. On the other hand, pre-incubation of the MDA-MB-231 cells with brefeldin A (a trans-Golgi protein trafficking inhibitor) had no effect on the E2-induced increase in VGSC amplitude, indicating that such trafficking ('externalisation') of VGSC was not involved. Finally, acute application of E2 decreased cell adhesion whilst the specific VGSC blocker tetrodotoxin increased it. Co-application of E2 and tetrodotoxin inhibited the effect of E2 on cell adhesion, suggesting that the effect of E2 was mainly through VGSC activity. Pre-treatment of the cells with PKI abolished the effect of E2 on adhesion, consistent with the proposed role of PKA. Potential implications of the E2-induced non-genomic upregulation of VGSC activity for BCa progression are discussed.

Molecular pharmacology of voltage-gated sodium channel expression in metastatic disease: clinical potential of neonatal Nav1.5 in breast cancer.

A variety of ion channels have been detected in cancer cells. In particular, upregulation of voltage-gated sodium channels (VGSCs) has been associated pathophysiologically with several strongly metastatic carcinomas. This review emphasises breast cancer. Inhibiting VGSC activity in a number of independent ways, using the highly selective tetrodotoxin (TTX), gene silencing and a blocking polyclonal antibody, suppressed a range of cellular behaviors, especially directional motility and invasion, integral to the metastatic cascade. Conversely, transfecting a VGSC into a weakly invasive human prostate cancer cell line significantly increased invasiveness. In vivo, also, VGSC expression has been correlated positively with metastatic status. It has been suggested, therefore (i) that VGSC upregulation is an early event in metastatic progression and (ii) that VGSC expression is a 'switch,' necessary and sufficient for engaging cancer cells in a highly invasive state. Importantly, where studied, mainly prostate and breast cancers, the dominant VGSC (Nav1.7 and Nav1.5, respectively) was found to be an embryonic/neonatal splice variant, consistent with the gene expression being "oncofoetal." In breast cancer, the molecular difference between the adult and neonatal isoforms of the VGSC/Nav1.5 is largest (31 base pairs, generating 7 amino acid differences). We propose that neonatal Nav1.5 is a novel marker with significant clinical potential for management of metastatic breast cancer and describe a number of approaches which may enable tumour-specific targeting. These include various small-molecule drugs, small-interfering RNA, monoclonal antibody and natural neurotoxins.

Alternative splicing of Nav1.5: an electrophysiological comparison of 'neonatal' and 'adult' isoforms and critical involvement of a lysine residue.

In developmentally regulated D1:S3 splicing of Nav1.5, there are 31 nucleotide differences between the 5'-exon ('neonatal') and the 3'-exon ('adult') forms, resulting in 7 amino acid differences in D1:S3-S3/S4 linker. In particular, splicing replaces a conserved negative aspartate residue in the 'adult' with a positive lysine. Here, 'neonatal' and 'adult' Nav1.5 alpha-subunit splice variants were stably transfected into EBNA-293 cells and their electrophysiological properties investigated by whole-cell patch-clamp recording. Compared with the 'adult' isoform, the 'neonatal' channel exhibited (1) a depolarized threshold of activation and voltage at which the current peaked; (2) much slower kinetics of activation and inactivation; (3) 50% greater transient charge (Na(+)) influx; (4) a stronger voltage dependence of time to peak; and (5) a slower recovery from inactivation. Tetrodotoxin sensitivity and VGSCbeta1-4 mRNA expression levels did not change. The significance of the charge-reversing aspartate to lysine substitution was investigated by mutating the lysine in the 'neonatal' channel back to aspartate. In this 'neonatal K211D' mutant, the electrophysiological parameters studied strongly shifted back towards the 'adult', that is the lysine residue was primarily responsible for the electrophysiological effects of Nav1.5 D1:S3 splicing. Taken together, these data suggest that the charge reversal in 'neonatal' Nav1.5 would (1) modify the channel kinetics and (2) prolong the resultant current, allowing greater intracellular Na(+) influx. Developmental and pathophysiological consequences of such differences are discussed.