hERG Gating Microdomains Defined by S6 Mutagenesis and Molecular Modeling

Abstract

Human ether-à-go-go–related gene (hERG) channels mediate cardiac repolarization and bind drugs that can cause acquired long QT syndrome and life-threatening arrhythmias. Drugs bind in the vestibule formed by the S6 transmembrane domain, which also contains the activation gate that traps drugs in the vestibule and contributes to their efficacy of block. Although drug-binding residues have been identified, we know little about the roles of specific S6 residues in gating. We introduced cysteine mutations into the hERG channel S6 domain and measured mutational effects on the steady-state distribution and kinetics of transitions between the closed and open states. Energy-minimized molecular models based on the crystal structures of rKv1.2 (open state) and MlotiK1 and KcsA (closed state) provided structural contexts for evaluating mutant residues. The majority of mutations slowed deactivation, shifted conductance voltage curves to more negative potentials, or conferred a constitutive conductance over voltages that normally cause the channel to close. At the most intracellular extreme of the S6 region, Q664, Y667, and S668 were especially sensitive and together formed a ringed domain that occludes the pore in the closed state model. In contrast, mutation of S660, more than a full helical turn away and corresponding by alignment to a critical Shaker gate residue (V478), had little effect on gating. Multiple substitutions of chemically distinct amino acids at the adjacent V659 suggested that, upon closing, the native V659 side chain moves into a hydrophobic pocket but likely does not form the occluding gate itself. Overall, the study indicated that S6 mutagenesis disrupts the energetics primarily of channel closing and identified several residues critical for this process in the native channel.

Concerted All-or-none Subunit Interactions Mediate Slow Deactivation of Human ether-à-go-go-related Gene K+ Channels

“…The R4A/R5A double mutation is located in the PAS-cap region, and R56Q is located in the PAS domain. The mutation F656I is located in a region of the S6 segment that contributes to formation of the "S6 bundle crossing," the intracellular activation gate (29). In this study, channels formed from individual subunits expressed and assembled naturally in oocytes are called X monomer , where X is either a WT or mutant subunit.…”

A recombinant N-terminal domain fully restores deactivation gating in N-truncated and long QT syndrome mutant hERG potassium channels

“…Previous work shows that the slow deactivation kinetics in wild-type hERG channels are apparently influenced by several different regions of the protein. Mutations in transmembrane regions, including the voltage-sensor (S1-S4) domains (12,25,26), the S4-S5 linker region that joins the voltage sensor to the pore (17,27), and the lower part of the S6 (28,29) change the kinetics of deactivation. Mutations at the amino terminal end of the eag domain (13,17), within the Per-Arnt-Sim (PAS) region of the eag domain (13,19) and in the C-terminal regions (30,31), accelerate deactivation gating.…”

Structural imperatives impose diverse evolutionary constraints on helical membrane proteins

“…The proteins used were rhodopsin (aligned to 1GZM), calcium ATPases (aligned to 1SU4, 1T5S, 1WPE, 1WPG, and 2AGV), and the voltage-gated potassium channels Kv1.1 and Kv3.3 (aligned to 2R9R). In addition, we made use of hand-curated alignments of KCNQ1 (32) and hERG (33) to portions of the 2R9R (Kv1.2) sequence and the diseasemutation database for these proteins compiled by Jackson and Accili (19) (see Table S2). …”