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

• Published in: The Journal of physiology Vol. 587; no. 11; pp. 2555 - 2566
• Main Authors: Stepanovic, Svetlana Z., Potet, Franck, Petersen, Christina I., Smith, Jarrod A., Meiler, Jens, Balser, Jeffrey R., Kupershmidt, Sabina
• Format: Journal Article
• Language: English
• Published: Oxford, UK The Physiological Society 01-06-2009; Blackwell Publishing Ltd; Blackwell Science Inc
• Subjects: Alanine; Amino Acid Sequence; Animals; Cardiovascular; CHO Cells; Computer Simulation; Conserved Sequence; Cricetinae; Cricetulus; ERG1 Potassium Channel; Ether-A-Go-Go Potassium Channels - antagonists & inhibitors; Ether-A-Go-Go Potassium Channels - chemistry; Ether-A-Go-Go Potassium Channels - genetics; Ether-A-Go-Go Potassium Channels - metabolism; Evolution, Molecular; Humans; Ion Channel Gating; Kinetics; Membrane Potentials; Models, Molecular; Molecular Sequence Data; Mutation; Oocytes; Phenethylamines - pharmacology; Potassium Channel Blockers - pharmacology; Protein Conformation; Structure-Activity Relationship; Sulfonamides - pharmacology; Transfection; Xenopus laevis
• ISSN: 0022-3751; 1469-7793
• DOI: 10.1113/jphysiol.2008.166694

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.