Neonatal Nav1.5 voltage-gated Na+ channel : regulation, electrophysiology and pharmacology

The overall aims of this PhD were (1) to evaluate the mechanisms controlling functional expression of neonatal Nav1.5 (nNav1.5), the predominant voltage-gated sodium channel (VGSC) subtype expressed in metastatic human breast cancer (BCa) cells, and (2) to characterize the electrophysiological and pharmacological properties of nNav1.5 compared with the adult Nav1.5 (aNav1.5) counterpart. Experiments were carried out under normoxic and hypoxic conditions. The Results chapter-1 demonstrates the hypoxic upregulation of functional VGSC (nNav1.5) expression in MDA-MB-231 human BCa cells, proposed to occur via positive feedback involving Na+ influx and activation of protein kinase A. Upregulation of nNav1.5, evident at mRNA, protein and signalling levels, led to significant augmentation of Matrigel invasion. The hypoxia-sensitive persistent Na+ current (INaP) played a significant role in the increased invasiveness. The Results chapter-2 shows that, compared to aNav1.5, nNav1.5 (i) exhibited depolarized activation, (ii) had slower activation/inactivation kinetics, (iii) allowed greater transient charge (Na+) influx, (iv) recovered from inactivation significantly more slowly, (v) exhibited greater use-dependent attenuation, and (vi) expressed larger INaP. Mutagenesis studies revealed the charge-reversing Asp211 (aNav1.5) to Lys211 (nNav1.5) switch to be predominantly responsible for these differences. Surprisingly, however, challenging the two splice variants with mono-, di- and trivalent cations generated only subtle differential effects in channel gating. The Results chapter-3 determines the sensitivities of nNav1.5 and aNav1.5 to various VGSC blockers. The effects of small-molecule drugs lidocaine, phenytoin, mexiletine, procaine, ranolazine and riluzole were similar. NESOpAb, a polyclonal antibody targeting nNav1.5, exhibited ~200-fold lower threshold and ~5-fold lower IC50 for inhibiting nNav1.5 vs. aNav1.5; the Lys/Asp211 residue was crucial to this difference. Spider toxins ProTx-II and HaTx were found to share a binding site in the nNav1.5/aNav1.5 splicing region, exhibiting ~25- and 5-fold selectivity for aNav1.5. Each Results chapter ends with a discussion and highlighting of clinical implications

Neonatal Nav1.5 : pharmacological distinctiveness of a cancer‐related voltage‐gated sodium channel splice variant

Background and Purpose Voltage-gated sodium (Na-V) channels are expressed de novo in carcinomas where their activity promotes invasiveness. Breast and colon cancer cells express the neonatal splice variant of Na(V)1.5 (nNa(V)1.5), which has several amino acid substitutions in the domain I voltage-sensor compared with its adult counterpart (aNa(V)1.5). This study aimed to determine whether nNa(V)1.5 channels could be distinguished pharmacologically from aNa(V)1.5 channels. Experimental Approach Cells expressing either nNa(V)1.5 or aNa(V)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 Na-V channels (lidocaine, procaine, phenytoin, mexiletine, ranolazine, and riluzole) could not distinguish between nNa(V)1.5 or aNa(V)1.5 channels. However, two tarantula toxins (HaTx and ProTx-II) and a polyclonal antibody (NESOpAb) preferentially inhibited currents elicited by either nNa(V)1.5 or aNa(V)1.5 channels by binding to the spliced region of the channel. Furthermore, the amino acid residue at position 211 (aspartate in aNa(V)1.5/lysine in nNa(V)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 nNa(V)1.5 channel can be distinguished pharmacologically from its nearest neighbour, aNa(V)1.5 channels. Thus, it may be possible to design low MW compounds as antimetastatic drugs for non-toxic therapy of nNa(V)1.5-expressing carcinomas