The N-terminal PAS domain directly regulates hERG channel gating in excised, inside-out patch-clamp fluorometry (PCF) recordings

Abstract

ABSTRACTHuman ERG is a voltage-activated, K-selective channel whose physiological role is to drive action potential repolarization in cardiac myocytes. To carry out its role in the heart, hERG has specialized gating (opening and closing) transitions that are regulated by the internal N-terminal PAS and C-terminal CNBH domains. The PAS and CNBHD domains interact directly and this interaction is required for the characteristic slow deactivation (closing) of hERG channels. But it is unclear whether PAS remains globally attached or dislodges from the CNBHD during gating. Interestingly the direct PAS-CNBHD interaction can be formedin transby co-expression of the PAS domain and hERG channels with a deleted PAS domain (hERG ΔPAS) in which the PAS domain is not attached to the channel with a peptide bond.In transexpression allows us to probe the biophysical mechanism for PAS domain attachment to the rest of the channel and in a broader sense allows us to test the mechanism for intracellular domain function in an ion channel, and test whether the PAS domain detaches or remains attached to the channel during gating. We report here that in excised patches from cells containing the hERG PAS domain fused to CFP and hERG ΔPAS channels fused to Citrine that 1) regulation of deactivation (slow deactivation conveyed by the PAS domain) was similar in on-cell and excised, inside-out patch configurations, 2) that regulation of deactivation persists for the lifetime of the patch (up to 30 minutes) in excised, inside-out mode, 3) that channel activity measured by activation of the channel with voltage pulses did not alter channel deactivation and 4) dual fluorescence and ionic current measurements using patch-clamp fluorometry (PCF) showed that only membrane patches containing PAS-CFP + hERG ΔPAS-Citrine had CFP and Citrine fluorescence and slow (regulated) deactivation, whereas control patches with hERG ΔPAS -Citrine had fast (unregulated) deactivation and Citrine fluorescence (but not CFP fluorescence) and control patches from hERG PAS-CFP - injected cells had neither currents nor CFP or Citrine fluorescence. Moreover, in PCF mode, we detected FRET from PAS-CFP + hERG ΔPAS-Citrine channels. Taken together, these results suggested that PAS - CFP remained associated with hERG ΔPAS-Citrine channels after membrane excision. We interpret these results to mean that the PAS domain was not dislodged from the channel despite mechanical (excised patch) and conformational (voltage) challenges and suggests that the PAS domain remained firmly attached to the hERG channel during gating.