The evolutionarily conserved residue A653 plays a key role in HERG channel closing

Abstract

Human ether-a-go-go-related gene (HERG) encodes the rapid, outwardly rectifying K + current I Kr that is critical for repolarization of the cardiac action potential. Congenital HERG mutations or unintended pharmaceutical block of I Kr can lead to life-threatening arrhythmias. Here, we assess the functional role of the alanine at position 653 (HERG-A653) that is highly conserved among evolutionarily divergent K + channels. HERG-A653 is close to the 'glycine hinge' implicated in K + channel opening, and is flanked by tyrosine 652 and phenylalanine 656, which contribute to the drug binding site. We substituted an array of seven (I, C, S, G, Y, V and T) amino acids at position 653 and expressed individual variants in heterologous systems to assess changes in gating and drug binding. Substitution of A653 resulted in negative shifts of the V 1/2 of activation ranging from −23.6 (A653S) to −62.5 (A653V) compared to −11.2 mV for wild-type (WT). Deactivation was also drastically altered: channels with A653I/C substitutions exhibited delayed deactivation in response to test potentials above the activation threshold, while A653S/G/Y/V/T failed to deactivate under those conditions and required hyperpolarization and prolonged holding potentials at −130 mV. While A653S/G/T/Y variants showed decreased sensitivity to the I Kr inhibitor dofetilide, these changes could not be correlated with defects in channel closure. Homology modelling suggests that in the closed state, A653 forms tight contacts with several residues from the neighbouring subunit in the tetramer, playing a key role in S6 helix packing at the narrowest part of the vestibule. Our study suggests that A653 plays an important functional role in the outwardly rectifying gating behaviour of HERG, supporting channel closure at membrane potentials negative to the channel activation threshold.

Identification of Functionally Critical Residues in the Channel Domain of Inositol Trisphosphate Receptors

“…This model also places these residues at the narrowest point where the helixes emerge into the cytosol, and therefore these residues could be located at the critical "gate" region of the channel. A cluster of adjacent hydrophobic residues is known to stabilize S6-S6 interaction interfaces in the gate of other channels (19,20,41). The S6 helix of IP 3 Rs and RyRs are highly conserved, and both Ile residues are present in all isoforms of this family of channels.…”

“…We initially selected to mutate highly conserved residues including two hydrophobic residues at the predicted membrane/cytosol interface and a series of positive and negatively charged residues in the distal portion of S6. Hydrophobic residues located at the narrowest point of the channel where the S6 helices cross over each other are known to play important roles in the gating of other ion channels (18,20,21,37). In a previous study we found that mutation of a candidate highly conserved residue (Phe-2592) had little effect on channel function (29).…”

SYMPOSIUM REVIEW: Gating of two pore domain potassium channels

“…As such, channel activity may either increase or decrease through the action of regulators which influence this gate. In hERG channels an equivalent alanine residue (A653) close to the glycine hinge forms tight contacts with residues in neighbouring subunits and supports channel closure at membrane potentials below the activation threshold (Stepanovic et al 2009).…”

The role of site-directed point mutations in protein misfolding

“…where f is a dimensionless quantity appropriately scaled with respect to k B T. Eqn (11) clearly indicates that the more negative the value of f the larger is the stability gap D representing a highly stable sequence in the target/native conformation. Hence, a sequence with a lower negative f value represents a marginally stable sequence.…”

“…The alignment of the homologous amino acid sequences has led to a consensus approach, which assumes that the conserved amino acid residues play a dominant role in implementing protein stability. [6][7][8][9][10][11] However, sequences generated by the consensus approach are not necessarily the most stable ones. 6 Random point mutation studies have revealed that some of these mutations dramatically affect the stability of a protein, which cannot be explained from the molecular principles of structural stability.…”