Abstract
Recent cryo-EM data have provided a view of the KCNH potassium channels molecular structures. However, some details about the cytoplasmic domains organization and specially their rearrangements associated to channel functionality are still lacking. Here we used the voltage-dependent dipicrylamine (DPA)-induced quench of fluorescent proteins (FPS) linked to different positions at the cytoplasmic domains of KCNH2 (hERG) to gain some insights about the coarse structure of these channel parts. Fast voltage-clamp fluorometry with HEK293 cells expressing membrane-anchored FPs under conditions in which only the plasma membrane potential is modified, demonstrated DPA voltage-dependent translocation and subsequent FRET-triggered FP quenching. Our data demonstrate for the first time that the distance between an amino-terminal FP tag and the intracellular plasma membrane surface is shorter than that between the membrane and a C-terminally-located tag. The distances varied when the FPs were attached to other positions along the channel cytoplasmic domains. In some cases, we also detected slower fluorometric responses following the fast voltage-dependent dye translocation, indicating subsequent label movements orthogonal to the plasma membrane. This finding suggests the existence of additional conformational rearrangements in the hERG cytoplasmic domains, although their association with specific aspects of channel operation remains to be established.
Highlights
Kv11.1 K+ channels are expressed in a variety of non-cardiac cells in which they play a key role in setting their electrical behaviour[1,2,3,4]
As shown with CNGA1, TRPV1 and AMPAr channels[16,23,24], we conclude that the observed variation in the quenching level due to changes in the position of the labels along the protein sequence and the additional detection of delayed fluorometric responses following the rapid DPA translocation, constitute a valuable method to gain some insights into the overall architecture of the hERG cytoplasmic domains, that could allow the detection of conformational protein rearrangements associated with channel functions that cause orthogonal movements of the labels toward the plasma membrane
We used fully functional channels labeled at different positions with a FP tag acting as a FRET donor and DPA, a non–fluorescent absorber whose distribution switches between the inner and outer membrane leaflets according to the membrane potential, that is able to act as an acceptor for cyan, green and yellow FP variants[15,16,17,18,22,23,24]
Summary
Kv11.1 (hERG, KCNH2) K+ channels are expressed in a variety of non-cardiac cells in which they play a key role in setting their electrical behaviour[1,2,3,4]. Since this rapid variation in the fluorometric signal is only due to the movement of the DPA probe, it is expected that the magnitude of the fluorescence change in response to a fixed magnitude voltage step can be used to determine the position of any static label with respect to the lipid bilayer acting as a reference plane. As shown with CNGA1, TRPV1 and AMPAr channels[16,23,24], we conclude that the observed variation in the quenching level due to changes in the position of the labels along the protein sequence and the additional detection of delayed fluorometric responses following the rapid DPA translocation, constitute a valuable method to gain some insights into the overall architecture of the hERG cytoplasmic domains, that could allow the detection of conformational protein rearrangements associated with channel functions that cause orthogonal movements of the labels toward the plasma membrane
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