Abstract

Today's scientific question is: What in the world is electricity?
And where does it go after it leaves the toaster?
          -- Dave Barry, "What is Electricity?"
The plasma membrane of a cell serves the vital function of partitioning the molecular contents of the cytoplasm from its external environment. These membranes are largely composed of amphiphilic lipids which self-assemble into highly insulating structures and thus present a large energy barrier to transmembrane ionic transport. However, the lipid matrix can be disrupted by a strong external electric field leading to an increase in transmembrane conductivity and diffusive permeability. These effects are the result of formation of aqueous pores in the membrane which also alter the electrical potential across the membrane. Current experimental techniques do not have the temporal and spatial resolutions required to measure the distribution and rapid evolution kinetics of these pores and are thus restricted to the measurement of steady-state or post-field responses.

The objective of this project is to develop a model to express the non-linear changes in electrical properties of a cell membrane in terms of size, number and dynamics of membrane pores. The model will incorporate measurements of transmembrane current and potential distribution from voltage clamp and imaging studies in calculating the evolution of electropores and changes in membrane electrical properties. Numerical implementation of this model will be used to study electroporation dynamics under a range of external electric field amplitudes. This approach will generate a framework linking theoretical concepts of membrane-field interaction with experimental observations of electroporation effects.

The double vaseline gap voltage clamp method (Chen and Lee, 1994) will be used to measure transmembrane current at different transmembrane potentials. This arrangement provides a spatially uniform transmembrane potential. The transmembrane current measured using this method can be isolated to the current due mainly to electroporation of the membrane. In addition, the voltage clamp method provides temporal resolution of the order of microseconds and the measurements can be made in the presence of an applied potential. These experimental measurements will be used to estimate model parameters.

This investigation will use optical imaging methods to measure the three-dimensional transmembrane potential distribution. Skeletal muscle cells stained with a potential-sensitive fluorescent dye will be imaged using a confocal microscope. The 3-D cell image will be reconstructed from a number of optical sections. A blind iterative deconvolution method will be developed to restore the optical sections. The deblurred optical sections will be used to visualize the steady-state transmembrane potential distribution. 3-D reconstruction of potentiometric dye distribution will be used to quantify the three-dimensional transmembrane potential distribution.

Changes in transmembrane potential and transmembrane conductivity accompany many instances of membrane-field interaction. These events include both useful and harmful effects of an electric field on biological systems. Examples of such phenomena include electroporation, nerve conduction and high-voltage injury. The proposed work will help in estimating the magnitude of changes in membrane electrical properties caused by electrical fields involved in such events.