Single-Cell Electrophysiology Reveals Verapamil’s Disruption of Bacterial Membrane Energetics

Verapamil, a clinically used calcium channel blocker, enhances the activity of several tuberculosis antibiotics, but its mechanism of action and physiological effects on bacteria remain unresolved. A central debate concerns whether verapamil primarily inhibits efflux pumps or disrupts membrane energetics. Here, we use Escherichia coli as a model system to quantify single-cell and population-level physiological responses to verapamil with high temporal resolution. Real-time measurements of the rotational speed of individual flagellar motors, a single-cell proxy for the proton motive force (PMF), reveal a heterogeneous response to verapamil: treated cells exhibit either a dose-dependent gradual decrease in PMF, or a rapid collapse of PMF. Although loss of the outer-membrane efflux channel TolC increases growth inhibition by verapamil, it does not alter the rapid PMF disruptions observed at the single-cell level, suggesting that efflux contributes to long-term susceptibility but not to the initial PMF disruption. Independent assays of population-level motility, pH, and membrane-integrity suggest that verapamil may selectively dissipate the electrical component of PMF while leaving intracellular pH largely unchanged. A minimal electrical circuit model captures both steady-state and dynamic behavior. Together, these findings demonstrate that verapamil rapidly and reversibly perturbs bacterial membrane energetics through a mechanism distinct from classical protonophores, helping to reconcile conflicting interpretations of its activity and clarifying how membrane effects may interact with efflux inhibition during antibiotic potentiation.