Mapping the hERG Channel Activation Gate Using SCAM

Abstract

hERG channels, important for cardiac repolarization, are susceptible to block by a wide range of therapeutic drugs intended for other targets. The S6 activation gate contributes to the efficacy of drug block, yet its location is unknown. We used cysteine mutagenesis and expression in Xenopus oocytes to identify S6 domain residues important in gating, and mapped these residues in energy-minimized homology models based on the crystal structures of MlotiK1 and Kv1.2 for the closed and open states, respectively (Wynia-Smith et al., J. Gen. Physiol., 2008). The predominant mutant phenotype was slowed channel closing and/or constitutive conductance at negative potentials. Focusing initially on cysteine mutations with wild-type behavior and thus little structural perturbation, we are using MTS reagents to identify residues selectively accessible in the open state. Mutants S654C, F656C, S660C and L666C span a region homologous to the activation gate in Shaker channels as well as a separate region predicted by our molecular models to form a closing gate. These mutants reacted with MTSET and displayed current inhibition that reversed upon addition of DTT to the bath. The control channel showed no reactivity to MTSET. S654C exhibited a progressive development of current block during pulses to positive voltages, suggestive of state-dependent block. Experiments under way to determine rates of state-dependent modification will further define the residues forming the occluding gate in hERG channels.