A New Method to Detect Intra- and Extracellular Conformational Changes in Ion Channels with High Temporal Resolution

Abstract

Ligand-gated ion channels (LGICs) and voltage-gated ion channels (VGICs) are membrane proteins that represent important drug targets. While electrophysiological techniques allow the distinction between open, closed and desensitized/inactivated states, it is not capable to directly detect conformational changes that might precede, accompany or follow the gating or ligand binding events of an ion channel. The simultaneous detection of current and emission from a fluorescent label in voltage-clamp fluorometry (VCF) has proven a powerful technique to overcome this limitation by allowing to monitor conformational rearrangements at the channel gate and in a protein domain of interest. However, ligand application in VCF is slow and limited to extracellular domains. Here, we developed a novel, ultrafast patch-clamp fluorometry (ufPCF) setup for use with both LGICs and VGICs. A combination of a digital mirror device, an emCCD camera and a piezo-driven perfusion tool enable us to achieve a time resolution of around 1 ms for ligand application and fluorescence detection. Importantly, ufPCF can be used in both outside-out and inside-out patches, irrespective of the position of fluorescent dye within the protein. For example, ufPCF allows easy access to the intracellular side of extracellularly labeled channels in inside-out patches, enabling the modulation of intracellular conditions while monitoring rearrangements in extracellular domains (and vice versa). Further, our ufPCF can be used with fluorescent unnatural amino acids, which can be introduced anywhere in the protein. Overall, we find this method to be highly versatile and to generally result in larger fluorescence changes compared to VCF. We anticipate ufPCF to significantly aid our understanding of the pharmacology and molecular function of both LGICs and VGICs and we intend to extend its use to studies using transporters and pumps.