Title: Analysis and Minimization of Ligand Concentration Errors at the Internal Face of Excised Patches
Abstract: Confocal patch-clamp fluorometry is a method combining patch-clamp techniques and confocal fluorescence microscopy for simultaneous investigation of channel activation and ligand binding in ion channels, as used for CNGA2 olfactory channels and HCN2 pacemaker channels. The fluorescent ligands fcAMP and fcGMP, and the reference DY647, used in these studies were negatively charged. Their fluorescence signals in the volume between patch membrane and pipette tip can depend significantly on the applied voltage if the conditions are not optimized, indicating a difference between actual ligand concentration at the patch and in the bath.