The PAS domain directly regulates hERG channel gating in patch-clamp fluorometry recordings

Ion channels are membrane proteins composed of distinct modular domains. One example is the voltage-activated human ERG (hERG) potassium channel, which has specialized gating (opening and closing) transitions that are regulated by an intracellular N-terminal Per-Arnt-Sim (PAS) domain. Direct interactions between the PAS domain and other intracellular domains are required for the characteristic slow deactivation (closing) that is a hallmark of hERG channels, but the mechanism for PAS domain regulation of gating remains unclear. Here, we asked whether the PAS domain remained globally attached or became detached from the channel during gating. To test this, we took advantage of a method to investigate direct hERG PAS domain interactions and regulation (in the absence of a peptide bond tethering the PAS domain to the rest of the channel) by forming channels from two separate parts, hERG PAS domains tagged with cyan fluorescent protein (PAS-CFP) and hERG subunits bearing a deleted PAS domain and tagged with Citrine fluorescent protein (hERG ΔPAS-Citrine). We report that the resulting PAS-CFP/hERG ΔPAS-Citrine channels had slow deactivation kinetics, similar to wild-type hERG, that remained the same in on-cell and excised, inside-out patches, and that persisted for the lifetime of the excised patch (30 min) and following voltage protocols to open and close the channels. In patch-clamp fluorometry recordings, membrane patches with PAS-CFP/hERG ΔPAS-Citrine channels had slow deactivation and robust CFP fluorescence, whereas negative control patches with hERG ΔPAS-Citrine channels had faster deactivation and negligible CFP fluorescence. We interpret these results to mean that the PAS domain did not globally detach but rather was globally attached to the rest of the channel during gating. Our results more broadly show an experimental framework for examining the mechanisms of direct intracellular domain regulation of ion channels.