This study demonstrates that NaKA, NHE3, and NHE1 are involved in cellular directedness during electrotaxis, which imply that Vmem is a spatiotemporal regulator between the leading edge and rear end. We suggest that NHE3 might be a cathode-specific sensor protein because there was an increased relocation of active NHE3 to the membrane protrusions, which was accompanied by an elevated pHi at those same sites that specifically occurred in cathode-directed cells, not in anode-directed ones, during electrotaxis. However, intracellular levels of active NHE3 and of PIP2 were increased in anode-directed cells while they were less in cathode-directed cells in comparison to randomly migrating control cells. This can result from the localized activity of NHE3 in the membrane protrusions of cathode-directed cells. Most of the data concerning NHE3 deal with its renal functions; however, few studies have demonstrated the interactions between NHE3 and cytoskeletal proteins and the related signaling pathways regarding cell motility. The actin-modifying agents cytochalasin and latrunculin have been found to inhibit epithelial NHE3 activity, whereas the housekeeping exchanger, NHE1, has been found to be virtually affected . Similarly, the cytoskeletal scaffolding protein ezrin has been shown to regulate NHE3 translocation in Caco-2 cells . Consistent with these studies, we demonstrate significant colocalizations of active NHE3 with the filopodia marker protein ß-actin on the leading-edge of both anode- and cathode-directed cells. The distinct patchy pattern of NHE3 may depend on the activity of NHE3 because only active NHE3 shows this pattern and, this activity seems to be specialized in the filopodia where ß-actin is present. This might be due to the close interaction of these two proteins during cell migration. Additionally, the quenched directedness in the presence of the NHE3-specific inhibitor S3226 supports the idea that NHE3 is involved in directional determination during electrotaxis. Moreover, we have shown both a physiological and mechanistic role for NHE3 in cellular motility and directedness via demonstrating the colocalization of active NHE3 outward proton fluxes accompanied ß-actin (decreased pHi) at the leading edge of directionally migrating cells.
In contrast to NHE3, neither the expression nor the distribution of NHE1 was affected in directionally migrating cells during electrotaxis. Although the pharmacological inhibition of NHE1 via the use of HOE 642 prevented the cells from perceiving their direction which they normally would during electrotaxis, this phenomenon might be due to one of its other crucial functions as a housekeeping protein, e.g. the regulation of intracellular pH and volume, or to its roles in cell migration [21–25]. Therefore, we conclude that NHE1 is not specifically involved in determining the direction of migration, but instead, maintains the overall pHi, volume, and osmotic balance, which is crucial to cellular physiology during directed cell motility.
The relatively hyperpolarized cell membrane on the leading edge versus the rear end of the cells, regardless of their direction, can be explained by the relocation of NaKA from the cytoplasm to the leading edge in both cathode- and anode-directed cells during electrotaxis. NaKA is a major regulator of Vmem and maintains the resting membrane potential by transporting two K+ ions in and three Na+ ions out of the cell. If the cell membrane is hyperpolarized on the leading edge, more NaKA needs to be recruited to those sites in order to reduce the excessive positive charges on the membrane and to bring Vmem level down to its resting potential. NaKA has already been shown to be exponentially activated as a function of the Vmem, which in turn leads to a hyperpolarized cell membrane [26, 27]. Independent of its role in ion transport, evidence exists for the involvement of NaKA in PI3K signaling and cell motility , in epithelial polarization and the suppression of invasion , and for its interaction with actin . Moreover, Vmem itself has already been shown to induce cytoskeletal modifications in F-actin, microtubules, and vinculin  and, in adherens junctions  in endothelial cells. Our study demonstrates that NaKA colocalizes with the focal adhesion marker protein vinculin at the leading edge of both anode- and cathode-directed cells, and its inhibition by oubain quenches cellular directedness. This suggests that NaKA is involved in focal adhesion turnover, and, hence, the inhibition of its activity interferes with directional cell motility. Moreover, considering that the cell membrane is only hyperpolarized on the front and depolarized only on the back, regardless of the migration direction, Vmem seems to maintain the persistent directedness rather than to perceive the direction by regulating the spatiotemporal NaKA mobility between the leading edge and the rear end of cells during directional cell motility. Additionally, both the total and phosphorylated NaKA levels were increased in cathode-directed (Calvaria) cells, which indicate that both the expression and the activity of the protein were elevated in those cells during electrotaxis. In contrast, in anode-directed (SaOS-2) cells, only the activity of the protein was increased, whereas the total level of the protein was not affected. These data indicate that NaKA activity is required in both anode- and cathode-directed cells.
Cellular speed was dramatically increased, especially in anode-directed cells, when NHE3 (300%) or NHE1 (500%) as well as FAK (500%, control) activity was inhibited. This suggests a motility suppressor role for both NHE3 and NHE1 in these cells during directional motility. An NHE inhibition-induced reduction in cell adhesion and motility has been reported in various cell types [33–35]. These studies state that the local extracellular pH levels at focal adhesion sites modulate the strength of the cell adhesion and thereby migration of the cells on a collagen I matrices. An increase in NHE1 activity (more protons) was also observed to result in tighter adhesion and decreased cell migration, whereas a lack of protons, due to low NHE1 activity, prevented adhesion and migration. Further studies should elucidate the exact mechanisms of interaction between NHE3 activity (pHi) and filopodia formation so as to understand its physio-mechanical roles in persistent, directional cell motility.
In contrast to NHE3 and NHE1, the pharmacological inhibition of NaKA using oubain divergently decreased and increased the speed of anode- and of cathode-directed cells by 71% and an increase by 67%, respectively. It has already been reported that independent of its role in ion transport, NaKA has various other functions in cells , including its role as a motility suppressor function in MDCK carcinoma cells ; however, the reason why NaKA activity divergently affects anode- and cathode-directed cells divergently in terms of cellular speed remains unknown and requires further investigation.