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.