Ion efflux measurement

Extravesicular buffer (15 ml) is placed in a disposable plastic beaker (Fisher). Use of such inexpensive beakers removes the need for tedious washing between trials. Approximately 75-100 ml of the liposome suspension is pipetted into the beaker and the electrode allowed to equilibrate until a stable baseline is achieved. The solution should be stirred at medium speed at all times and no bubbles should adhere to the electrode tips. Once the baseline has stabilized, 2 ml of a 70 mM methanol stock of the K+-specific ionophore valinomycin is added to the beaker. Valinomycin induces the formation of a Nernst diffusion potential of —135 mV, negative inside. The ionophore may be omitted if no membrane potential is desired. The electrode is again allowed to equilibrate following valinomycin addition. The Bcl-2 family protein is then added to a concentration that will induce release of 50-80% of the total encapsulated Cl~ and induce a response having a slope between 30 and 60° which facilitates accurate calculation of the efflux rate. After the protein-induced efflux has levelled off, Triton X-100 is added to the beaker to a final concentration of 0.1%. This detergent addition lyses the vesicles and releases any residual chloride, allowing the total amount of chloride encapsulated to be determined. Examples of chloride-efflux induced by Bcl-2 family proteins, as measured by these methods, can be found in previous publications (36,38,48).

At the end of the measurements, a calibration curve is produced by additions of known amounts of KC1. The KC1 is added in the presence of an aliquot of liposomes, so that the background CI" concentration is similar. KC1 is added in progressively increasing amounts until the chart recorder deflection equals the maximum observed by Triton X-100 lysis of the vesicles.

For colicin molecules, where the molecularity of the channel is known to be 1 (58), a specific CI" efflux rate can be calculated by determining the ratio of colicin-induced CI" release to the total encapsulated CI". This value is then divided by the number of colicin molecules present (assuming that each molecule forms one channel) and the quotient divided by the time taken for the efflux to occur. Since the molecularity of the Bcl-2 protein family channels is still unclear, this rate calculation is not as straightforward. For purposes of comparison between trials, this laboratory simply determines the slope of the initial response.

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