Abstract
The HERG K+ channel has very unusual kinetic behavior that includes slow activation but rapid inactivation. These features are critical for normal cardiac repolarization as well as in preventing lethal ventricular arrhythmias. Mutagenesis studies have shown that the extracellular peptide linker joining the fifth transmembrane domain to the pore helix is critical for rapid inactivation of the HERG K+ channel. This peptide linker is also considerably longer in HERG K+ channels, 40 amino acids, than in most other voltage-gated K+ channels. In this study we show that a synthetic 42-residue peptide corresponding to this linker region of the HERG K+ channel does not have defined structural elements in aqueous solution; however, it displays two well defined helical regions when in the presence of SDS micelles. The helices correspond to Trp585-Ile593 and Gly604-Tyr611 of the channel. The Trp585-Ile593 helix has distinct hydrophilic and hydrophobic surfaces. The Gly604-Tyr611 helix corresponds to an N-terminal extension of the pore helix. Electrophysiological studies of HERG currents following application of exogenous S5P peptides show that the amphipathic helix in the S5P linker interacts with the pore region of the channel in a voltage-dependent manner.
Highlights
The HERG K؉ channel has very unusual kinetic behavior that includes slow activation but rapid inactivation
HERG is a member of the family of voltage-gated Kϩ channels (VGK)1 that contain six transmembrane domains, denoted by S1–S6, and a pore helix that is interposed between S5 and S6
The HERG Kϩ channel functions as an inward rectifier, i.e. it passes little current at depolarized potentials but large currents during the terminal repolarization phase of the cardiac action potential [13, 14]. This rapid inactivation is critical for the role of the channel in suppressing arrhythmias initiated by ectopic electrical excitation [11, 15]
Summary
Peptide Preparation—The peptides were synthesized on a 0.50-mmol scale using O-(Benzotriazol-1-yl)-N,N,NЈ,NЈ-tetramethyluronium hexafluorophosphate activation of Boc-amino acids with in situ neutralization chemistry, as previously described [37]. The syntheses were performed on Boc-Tyr(2BrZ)-OCH2-Pam resin using standard amino acid side chain protection, except that methionine residues at positions 5 and 10 were replaced by the isosteric norleucine residue to prevent adventitious oxidation of the peptide. This step is necessary to stabilize the synthetic peptide and is not expected to affect the peptide conformation [37]. Of the 1600 structures generated in DYANA, 40 of the “best” structures, with the lowest NOE violations, were chosen for refinement using the standard simulated annealing script in CNS [45] In this refinement process, the high temperature dynamics and cooling cycle were performed in Cartesian space. Where A and C are constants, obs is the observed single exponential time constant measured from the rate of change in current following addition of the peptide, deact is the time constant of deactivation (estimated from the single exponential fit to the current recorded in the absence of peptide), and on is the apparent time constant for peptide binding, i.e. on ϭ 1/
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