During voltage-dependent activation in Shaker channels, four arginine residues in the S4 segment (R1-R4) cross the transmembrane electric field. It has been proposed that R1-R4 movement is facilitated by a "gating charge transfer center" comprising a phenylalanine (F290) in S2 plus two acidic residues, one each in S2 and S3. According to this proposal, R1 occupies the charge transfer center in the resting state, defined as the conformation in which S4 is maximally retracted toward the cytoplasm. However, other evidence suggests that R1 is located extracellular to the charge transfer center, near I287 in S2, in the resting state. To investigate the resting position of R1, we mutated I287 to histidine (I287H), paired it with histidine mutations of key voltage sensor residues, and determined the effect of extracellular Zn 2+ on channel activity. In I287H+R1H, Zn 2+ generated a slow component of activation with a maximum amplitude (A slow,max) of ~56%, indicating that only a fraction of voltage sensors can bind Zn 2+ at a holding potential of -80 mV. A slow,max decreased after applying either depolarizing or hyperpolarizing prepulses from -80 mV. The decline of A slow,max after negative prepulses indicates that R1 moves inward to abolish ion binding, going beyond the point where reorientation of the I287H and R1H side chains would reestablish a binding site. These data support the proposal that R1 occupies the charge transfer center upon hyperpolarization. Consistent with this, pairing I287H with A359H in the S3-S4 loop generated a Zn 2+-binding site. At saturating concentrations, A slow,max reached 100%, indicating that Zn 2+ traps the I287H+A359H voltage sensor in an absorbing conformation. Transferring I287H+A359H into a mutant background that stabilizes the resting state significantly enhanced Zn 2+ binding at -80 mV. Our results strongly support the conclusion that R1 occupies the gating charge transfer center in the resting conformation. © 2011 Lin et al.
CITATION STYLE
Lin, M. chin A., Hsieh, J. Y., Mock, A. F., & Papazian, D. M. (2011). R1 in the Shaker S4 occupies the gating charge transfer center in the resting state. Journal of General Physiology, 138(2), 155–163. https://doi.org/10.1085/jgp.201110642
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