A method for the rapid exchange of solutions bathing excised membrane patches

In this communication we describe a technique for rapidly exchanging solutions bathing excised membrane patches, and present examples of its implementation using both outside-out and inside-out patches. The ability to make step changes in the concentration of channel-activating ligands (e.g., acetylcholine, calcium) offers a novel and direct means of measuring kinetic processes in the 10-100-ms range. The responses to step ligand concentration changes are well suited to ensemble variance analysis, yielding estimates of the number of channels in a patch, and testing assumptions of channel independence and homogeneity. Kinetic analysis of the pseudomacroscopic currents obtained by averaging large numbers of responses can be compared and correlated with analysis of the microscopic behavior of single channels, using the same membrane patch for both approaches. Practical and theoretical limitations associated with the method are briefly discussed.