Introduction

Planar bilayer membranes formed over a small aperture in a partition separating two compartments represent a versatile platform for studies of membrane ion channels (1,2,3), membrane fusion (4), as well as the development of biosensors (5). The method of the planar bilayers allows controlling precisely the composition of solutions on both sides of the bilayer, as well as the composition of the bilayer itself. Ion channels from intracellular organelles (such as sarcoplasmic reticulum or mitochondria) not directly accessible to the patch-clamp technique can be incorporated into the bilayer and investigated under precisely defined experimental conditions. The limiting step in the effectiveness of the method is its high demand of labor skills and attention. Several promising solutions to the problem of aperture fabrication for low-noise recordings (1,6,7) and reduction of the time required to form lipid-bilayer have been introduced (8). Despite the continuing progress in bilayer technology, software utilities for online monitoring of bilayer formation similar to the tools aiding in patch-clamp technique (e.g., pClamp, www.moleculardevices.com; PULSE, www.heka.com; PCP Analyzer) (9) are not commonly available. Monitoring of bilayer formation is done typically with saw-tooth or triangular voltage stimulation (1,8), since the amplitude of the resulting square wave current response is proportional to the value of bilayer capacitance. Users of PULSE and X-CHART (www.heka.com) are able run the protocol and macro files developed originally for the two-electrode voltage clamp measurement of membrane capacitance using the paired-ramps stimulus protocol (10); however there is no such opportunity for users of other popular systems, such as the pClamp and Digidata 132× family of data acquisition systems (www.moleculardevices.com). To fill this gap in the software support of bilayer experiments, we have developed a multipurpose software tool that features real-time, high-resolution analysis of capacitance and resistance of lipid bilayer as well as other useful automated functions, such as sound alerts informing about bilayer formation, thinning, and breakage, and automatic zeroing of voltage offsets prior to membrane formation. This software, called bilayer lipid membrane (BLM) Analyzer, integrates current responses within specified time segments (11) to provide high-resolution capacitance estimation in a way similar to the previous report using the unipolar voltage stimulus consisting of two consecutive ramps of inverse slope (10). We have developed this idea further and extended the algorithm to use with bipolar saw-tooth voltage stimulus, allowing high-resolution measurement of capacitance, resistance, and leak current at the same time with the additional advantage of improved rejection of power line interference.

Materials and Methods

Principle of the Measurement

The measurement chamber for bilayer experiments with small a aperture in the separating wall can be represented as parallel combination of capacitance C_B and resistance R_B (Figure 1A). Prior to the formation of the bilayer, C_B corresponds to the small parasitic capacitance of the separating wall, and R_B corresponds mostly to the resistance of the aperture filled with electrolyte solution (3). After formation of the bilayer, C_B represents the capacitance of the bilayer, and R_B represents the resistance of the bilayer. The access resistance, which includes the resistance of electrodes, salt bridges, bath solution, and solution within the aperture, is negligible compared with bilayer resistance (3) and can be ignored when calculating C_B and R_B. The process of bilayer formation can be monitored by measuring the current i_B(t) flowing through the chamber during the bipolar symmetrical saw-tooth voltage stimulation v(t) (Figure 1B). Since the current through the capacitance is i_C=C_B×dv/dt, while the current through resistance is simply i_R=v/R_B, the resulting i_B(t) consists of the square wave current component with amplitude 4UC_B/T_S flowing through the capacitance and the saw-tooth current component with amplitude U/R_B flowing through the resistance, where U stands for the amplitude of the voltage stimulus and T_S stands for the stimulus period (Figure 1B). Integration of the current response i_B(t) within specific time segments provides charges Q_A⁺, Q_A⁻, Q_B⁺, and Q_B⁻ (Figure 1B). The value of each of these charges is simply the area of trapezoid (Figure 1B), which can be calculated as average current within the given time segment multiplied by the width of the time segment: