Voltage Clamp Measurement of Electroporation Current

In 1786 Luigi Galvani supposedly was performing experiments with a machine in the company of friends, when, by chance, one member of the party idly probed with a knife the nerves of the thigh of a skinned frog to be used for soup. As the muscles of the frog leg suddenly and unexpectedly contracted, Galvani's wife noted that a spark had been produced by the electrical machine and "fancied that there was an agreement in point of time." -Encyclopedia Britannica
Specific Aim: The increase in transmembrane current in the presence of an external electric field and its relaxation following the removal of the field are caused by changes in pore population. Thus, transmembrane current under an applied field can be used to estimate the factors related to pore population and dynamics. When a cell is placed in an electric field, both transmembrane current and transmembrane potential change dynamically. However, one unknown can be removed from the measurement process by holding the transmembrane potential constant during the measurement of transmembrane current. Thus, transmembrane current will be measured using a voltage clamp technique over a range of transmembrane potentials. Pore creation rate dependence on transmembrane potential will be estimated from these measurements for use in the proposed model.

Experimental Methods and Materials: A series of membrane electroporation current measurements will be performed using the voltage clamp technique. These measurements will be used to determine the relation between transmembrane potential and transmembrane current. The voltage clamp technique involves clamping a constant voltage across a cell membrane and simultaneously measuring the transmembrane current. This makes it possible to calculate changes in membrane conductance during the pulse.

Voltage Clamp Configuration: An improved double vaseline gap voltage clamp (Chen and Lee, 1994) will be used to measure transmembrane current as a function of transmembrane potential (Fig. 8). The experimental chamber will be divided into three partitions. The middle partition, central pool (CP), will be 300 m in width while each adjacent partition, end pool (EP), will be 100 m wide. An isolated muscle cell will be mounted in the notches of the two partitions. The cell will be held in place, spanning the central pool, by two Delrin clips attached to the bottom of the two end pools using high vacuum grade stop-cock grease. Thin vaseline seals and two glass cover slips will be used to electrically isolate the three pools from each other. The two end pools are electrically connected and are constrained by a command pulse (V). The command pulse is used in a feedback mechanism to adjust the injected current (I) to maintain or clamp the transmembrane potential at a prescribed level.

Measurement Protocol: A Total Clamp 8800 (Dagan Co., Minneapolis, MN) will be used in all the voltage clamp measurements. The control commands will be transmitted by an IBM personal computer-based digital-to-analog converter. The shape, magnitude and duration of the electrical pulses will be specified using a software interface. The transmembrane current will be filtered at 3kHz by an electronic filter (902LPF) and digitized by a digital oscilloscope (Tektronix 11401, Beaverton, OR). Measurements will be made for a series of 4 to 800 msec pulses clamping the membrane at potentials ranging from -140 to -400 mV.

Figure 5a: Transmembrane current in response to a series of square pulses of 8 msec duration measured using the voltage clamp setup. The transmembrane potential in these experiments ranged from -100 mV to -390 mV in steps of -10 mV. The membrane current profiles (bottom) were obtained from the raw data (top) by noise removal using the wavelet shrinkage method. The post-field data was fit to an exponential function to obtain the relaxation time constant (t).

Preliminary Studies: The objective of voltage clamp studies was to measure transmembrane current under constant transmembrane potentials and to incorporate these measurements into the theoretical model. Transmembrane current under a constant transmembrane potential was measured (Fig. 5a,Fig. 5b) using a double vaseline gap voltage clamp technique (Chen and Lee, 1994). This technique has the advantage that the transmembrane potential can be maintained at a constant value without significant spatial variations. In addition, using channel blockers and substituting certain ions, the transmembrane current can be isolated to electroporation current.

Figure 5b: Transmembrane current in response to a series of square pulses of 48 msec duration measured using the voltage clamp setup. The transmembrane potential in these experiments ranged from -140 mV to -230 mV in steps of -10 mV. The membrane current profiles (bottom) were obtained from the raw data (top) by noise removal using the wavelet shrinkage method. The post-field data was fit to an exponential function to obtain the relaxation time constant ().

Single muscle cells from the semitendinosus of the English frog rana temperoria were used in these measurements. The spacing between the clamps holding the cell was 300 m, which is small compared to the space constant of the cell thus ensuring relatively uniform transmembrane potential along the length of the cut-cell. The cell was held at -90 mV holding potential prior to applying the stimulation pulses. Figure 5a shows a time profile of the transmembrane current in response to different transmembrane potential pulses. A series of 8 msec pulses in the range of -100 to -390 mV were used to study the evolution of transmembrane current during the pulse and relaxation following the pulse. The wavelet shrinkage method of noise removal (Donoho and Johnstone, 1992) was used in extracting the signal (Fig. 5a,Fig. 5bbottom) from the measured data (Fig. 5a,Fig. 5btop). Briefly, the signal was recovered by a three-step process. The wavelet transform of the recorded data was computed, a soft threshold was applied on the resulting wavelet coefficients and finally, the inverse wavelet transform was computed to obtain the restored signal.

The results of Fig. 5a indicate that the current did not reach steady-state within 8 msec in the range of potentials studied. Thus, longer pulses at smaller pulse amplitudes were studied. Figure 5b shows the transmembrane current profiles in response to 48-msec pulses in the range of -140 to -230 mV. It is seen that only small amplitude pulses (<200 mV) show steady-state behavior. Results for transmembrane potentials higher than 200 mV indicate that the balance between pore expansion and contraction is not reached within the pulse duration. Longer transmembrane potential pulses are required to measure the steady-state response of transmembrane current. Figures 5a and 5b also list the post-field relaxation time constant () for different transmembrane potentials. The relaxation time constant information will be used in the theoretical model to estimate the pore destruction rate.