R1 in the Shaker S4 Occupies the Gating Charge Transfer Center in the Resting State

Abstract

During Shaker activation, R1-R4 in S4 cross the transmembrane field. Recently, Tao et al proposed that F290 and E2 in S2 and D3 in S3 constitute a ‘gating charge transfer center’ occupied by R1 at rest (Science 328:67-73, 2010). In contrast, previous evidence suggests that R1 is extracellular to F290, near E1 and I287 in S2, at rest. We investigated the resting location of R1 using engineered Zn2+ binding sites in which I287H was paired with another histidine mutation. Importantly, binding sites involving I287H are located extracellular to the charge transfer center. In I287H+F324H, Zn2+ slowed opening, increased the delay before opening, and shifted the voltage dependence of the delay in the depolarized direction. These results indicate that Zn2+ binds to and stabilizes the resting state. In I287H+R1H, Zn2+ generated a slow component of activation. Its amplitude (Aslow) was ∼55% in saturating Zn2+, suggesting that only some voltage sensors can bind Zn2+ at −80 mV. The maximal Aslow decreased after depolarizing and hyperpolarizing prepulses. The decline of Aslow after negative prepulses supports the idea that R1 moves inward and occupies the charge transfer center upon hyperpolarization. Consistent with this, pairing I287H with A359H, located in the S3-S4 loop, generated a binding site. In high Zn2+, a slow component of activation accounted for >80% of the kinetics, indicating that Zn2+ traps the voltage sensor in an absorbing conformation. We transferred I287H+A359H into the F290W+R1K+K5R background, which stabilizes the resting state. Zn2+ slowed activation kinetics, which were well fitted by one component in low Zn2+. Therefore, stabilizing the resting state allows most I287H+A359H voltage sensors to bind Zn2+ at −80 mV. We conclude that R1 occupies the gating charge transfer center in the resting conformation. NIH-R01GM43459 (DMP)