Cell membrane response to electrical stress at nanosecond and nanometer resolution

Pulsed electric fields (PEFs) are widely used in medical applications ranging from neuromodulation and neuromuscular stimulation to tissue and cancer ablation. The primary effect of PEF treatments is charging of the cell plasma membrane for cell activation. However, excessive charging disrupts the membrane through a process known as electroporation or electropermeabilization. Electroporation can be caused by routine electrostimulation; it is a significant adverse effect of defibrillation and a pathogenic factor in electrical trauma. Conversely, controlled electropermeabilization enables gene electrotransfer, pulsed field ablation for atrial fibrillation, cancer ablation, and electrochemotherapy. PEF treatments must be tailored to each application, grounded in fundamental knowledge of how the cell membrane responds to electrical stress. Understanding the kinetics of membrane charging and relaxation, lesion formation and repair mechanisms, their dependence on PEF parameters and cell physiology will create a framework for fine-tuning PEF effects – either for efficient stimulation without damage, or for effective ablation with minimal neuromuscular activation. Our team has long been involved in studying electropore properties and impact on cell function, excitability, and survival. We recently advanced the field by pioneering dynamic imaging of single electropores in live cells and by developing pulsed laser strobe microscopy to capture membrane charging and relaxation kinetics on the nanosecond timescale. This research proposal will explore membrane response to PEF at the single-pore level and with nanosecond resolution, to systematically characterize the primary mechanisms of complex electropermeabilization phenomena. We will design and validate novel mechanisms-based PEF protocols for medical applications. Aim 1: Quantify the formation of membrane lesions (diffuse permeabilization, transient and persistent focal pores) with respect to PEF duration (nano- to milliseconds) and the electric field strength (0.1-10 kV/cm). Compare effects of single PEFs and PEF trains at 10–90% duty cycles. Correlate lesion types with membrane charging and relaxation kinetics in cells of different shape and excitability. Characterize membrane charging by MHz compression of nsPEF bursts and bipolar cancellation mechanisms. Aim 2: Characterize the lifecycle, permeability, and current-voltage function of single electropores and their dependence on cell physiology, environment, and PEF protocols. Investigate electropore association with lipid rafts, voltage-gated channels, and structural proteins. Analyze whether membrane proteins form electropores and/or contribute to electropore structure and longevity. Test the electrodeformation mechanism of electropermeabilization. Aim 3. Examine the protective role of the adaptive electropore conductance and analyze the underlying mechanisms. Design novel nsPEF protocols to control electropermeabilization. Quantify cellular protection conferred by the activation of voltage-gated ion channels, lanthanide ions, and poloxamers. Primary membrane effects of PEF determine diverse downstream physiological effects and cell survival. A detailed understanding of these early events is key to developing effective treatments and minimizing adverse effects. Project Number: 1R01GM160649-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Andrei Pakhomov | Institution: OLD DOMINION UNIVERSITY, NORFOLK, VA | Award Amount: $320,000 | Activity Code: R01 | Study Section: Biochemistry and Biophysics of Membranes Study Section[BBM] View on NIH RePORTER: https://reporter.nih.gov/project-details/11344427