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Structural and Functional Role of the Extracellular S5-P Linker in the HERG Potassium Channel

Jie Liu, Mei Zhang, Min Jiang and Gea-Ny Tseng

Department of Physiology, Virginia Commonwealth University, Richmond, VA 23298

Address correspondence to Gea-Ny Tseng, Department of Physiology, Virginia Commonwealth University, 1101 E. Marshall Street, Richmond, VA 23298. Fax: (804) 828-7382; E-mail: gtseng{at}hsc.vcu.edu

C-type inactivation in the HERG channel is unique among voltage-gated K channels in having extremely fast kinetics and strong voltage sensitivity. This suggests that HERG may have a unique outer mouth structure (where conformational changes underlie C-type inactivation), and/or a unique communication between the outer mouth and the voltage sensor. We use cysteine-scanning mutagenesis and thiol-modifying reagents to probe the structural and functional role of the S5-P (residues 571–613) and P-S6 (residues 631–638) linkers of HERG that line the outer vestibule of the channel. Disulfide formation involving introduced cysteine side chains or modification of side chain properties at "high-impact" positions produces a common mutant phenotype: disruption of C-type inactivation, reduction of K⁺ selectivity, and hyperpolarizing shift in the voltage-dependence of activation. In particular, we identify 15 consecutive positions in the middle of the S5-P linker (583–597) where side chain modification has marked impact on channel function. Analysis of the degrees of mutation-induced perturbation in channel function along 583–597 reveals an -helical periodicity. Furthermore, the effects of MTS modification suggest that the NH₂-terminal of this segment (position 584) may be very close to the pore entrance. We propose a structural model for the outer vestibule of the HERG channel, in which the 583–597 segment forms an -helix. With the NH₂ terminus of this helix sitting at the edge of the pore entrance, the length of the helix (20 Å) allows its other end to reach and interact with the voltage-sensing domain. Therefore, the "583–597 helix" in the S5-P linker of the HERG channel serves as a bridge of communication between the outer mouth and the voltage sensor, that may make important contribution to the unique C-type inactivation phenotype.

Key Words: rapid delayed rectifier K channel • LQT2 • cysteine-scanning mutagenesis • oocyte expression

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