| hERG K+ 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, little is known about the roles of specific S6 residues in gating. My thesis work aimed to determine the location of the hERG activation gate and to elucidate the contribution of individual amino acids to the structure of the gate region. Cysteine mutations were introduced into the hERG channel S6 domain and mutational effects on the steady-state distribution and kinetics of transitions between the closed and open states were measured. Energy-minimized molecular models based on the rKv1.2 crystal structure (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. Multiple substitutions of chemically distinct amino acids were made at position V659, which is one position away by homology from the Shaker gate. Kinetic and molecular modeling results suggested that, upon closing, the native V659 side chain moves into a hydrophobic pocket of defined size but likely does not form the occluding gate itself. 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. Further mutagenesis at position Q664 resulted in multiple channels showing constitutive conductance over negative potentials, consistent with a role in gating. Investigation of S660C channels in excised macropatches showed a dramatic current decrease upon excision into the oxidizing bath environment. This is consistent with formation of a spontaneous disulfide bond which blocks current flow and suggests S660 side chains contribute to the gate structure. Together, these results suggest the hERG activation gate may be doubly closed in nature, formed by the side chains of S660 and Q664. |
