Voltage-gated Na⁺ channels are expressed in many cancer types (1), and in breast cancer Na_v1.5 expression is associated with increased metastasis and poorer survival (2). Na_v1.5 activity has been shown to increase invasion of breast cancer cells by reducing the extracellular pH. This is dependent on Na_v1.5 activity increasing H⁺ extrusion through the Na⁺/H⁺ exchanger NHE1 (3). The mechanism by which Na_v1.5 increases H⁺ release into the tumour microenvironment is unclear. This study investigated whether Na_v1.5 activity increases the rate of glycolysis, since an increased influx of Na⁺ means that more ATP is needed to power Na⁺/K⁺ ATPase to maintain intracellular [Na⁺] at steady state.

In MDA-MB-231 breast cancer cells at an extracellular pH of 6.0 (as can be found in solid tumours), the steady state (“persistent”) current through Na_v1.5 channels at the resting membrane potential of -18.9 mV (4) was 10.3 ± 2.2% of the maximal transient Na⁺ current, which was -9.19 ± 1.28 pA/pF. For an average cell with capacitance 26.0 ± 2.2 pF, this equated to a persistent inward Na⁺ current of -24.6 ± 3.4 pA.

We showed Na⁺ removal from the cell via Na⁺/K⁺ ATPase relies on glycolysis for ATP in this model, because inhibiting glycolysis with iodoacetate increased intracellular [Na⁺], as measured by the ratiometric indicator SBFI-AM (n = 6, p <0.01, one sample t test), whereas inhibiting oxidative phosphorylation with oligomycin did not change the intracellular [Na⁺] (n = 6, p = 0.62, one sample t test).

Using a Seahorse analyzer, we then showed that Na_v1.5 activity increased glycolysis, measured as the extracellular acidification rate, by 9.8 ± 1.7 mpH units/minute (n = 3, p < 0.0001, 2-way ANOVA), whereas Na_v1.5 activity did not change oxidative phosphorylation, measured as the oxygen consumption rate (n = 3, p = 0.99, 2-way ANOVA).

The rate of Na_v1.5-dependent H⁺ production was estimated by assuming that all Na⁺ entering via Na_v1.5 was removed by the Na⁺/K⁺ ATPase, and this was powered by ATP from glycolysis. It was assumed that H⁺ produced via ATP hydrolysis and glycolysis was then removed by NHE1. The estimated rate of pH change in a Seahorse analyzer well was 1.3 mpH units/minute, which was in the same order of magnitude as the measured rate of pH change.

These results indicate that Na⁺ channel activity in cancer cells can increase glycolytic respiration which acidifies the tumour microenvironment, and this may explain how Na_v1.5 increases invasion in breast cancer.