Slow activation and rapid C-type inactivation produce inward rectification of the current-voltage relationship for human ether-a'-go-go-related gene (hERG) channels. To characterize the voltage sensor movement associated with hERG activation and inactivation, we performed an Ala scan of the 32 amino acids (Gly 514 -Tyr 545 ) that comprise the S4 domain and the flanking S3-S4 and S4 -S5 linkers. Gating and ionic currents of wild-type and mutant channels were measured using cut-open oocyte Vaseline gap and two microelectrode voltage clamp techniques to determine the voltage dependence of charge movement, activation, and inactivation. Mapping the position of the charge-perturbing mutations (defined as ͦ⌬⌬Gͦ > 1.0 kcal/mol) on a threedimensional S4 homology model revealed a spiral pattern. As expected, mutation of these residues also altered activation. However, mutation of residues in the S3-S4 and S4 -S5 linkers and the C-terminal end of S4 perturbed activation (ͦ⌬⌬Gͦ > 1.0 kcal/mol) without altering charge movement, suggesting that the native residues in these regions couple S4 movement to the opening of the activation gate or stabilize the open or closed state of the channel. Finally, mutation of a distinct set of residues impacted inactivation and mapped to a single face of the S4 helix that was devoid of activation-perturbing residues. These results define regions on the S4 voltage sensor that contribute differentially to hERG activation and inactivation gating. The human ether-a'-go-go-related gene (hERG)1 channels are primarily expressed in the brain and the heart (1) but are also up-regulated in tumors from a variety of tissues (2). In the heart, hERG channels conduct the rapidly activating, delayed rectifier potassium current, I Kr (3, 4). Unlike most voltagegated K ϩ (Kv) channels, the fully activated current-voltage relationship of hERG channels exhibits inward rectification, a property that limits efflux of K ϩ during the plateau phase of the cardiac action potential.Inward rectification of hERG channels results from a rapid and voltage-dependent C-type inactivation that proceeds at a rate much faster than activation. At membrane potentials between Ϫ20 and ϩ20 mV, channels activate over hundreds of milliseconds but inactivate in milliseconds (5-7). The voltage dependence of hERG inactivation is shifted nearly Ϫ65 mV relative to channel activation (V 0.5INACT ϭ Ϫ85 mV, V 0.5ACT ϭ Ϫ20 mV). A single mutation (S631A) in the P-loop of the outer pore causes a ϩ100 mV shift in the voltage dependence of inactivation, but no shift in activation compared with wild-type (WT) channels (6, 8). Furthermore, external cations have differential effects on the voltage dependence of activation and inactivation (9 -11). Together, these findings led to the suggestion that distinct voltage-sensing mechanisms underlie activation and inactivation gating in hERG channels.The highly charged S4 transmembrane helix functions as the primary voltage sensor in voltage-gated channels. Neutralization of basic S4 residues shifts the vol...