Gating mechanism of Kv11.1 (hERG) K+ channels without covalent connection between voltage sensor and pore domains

Pilar De La Peña,Francisco Barros,Pedro Domínguez

doi:10.1007/s00424-017-2093-9

Pilar De La Peña, Francisco Barros + Show 1 more

Open Access

https://doi.org/10.1007/s00424-017-2093-9

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Abstract

Kv11.1 (hERG, KCNH2) is a voltage-gated potassium channel crucial in setting the cardiac rhythm and the electrical behaviour of several non-cardiac cell types. Voltage-dependent gating of Kv11.1 can be reconstructed from non-covalently linked voltage sensing and pore modules (split channels), challenging classical views of voltage-dependent channel activation based on a S4–S5 linker acting as a rigid mechanical lever to open the gate. Progressive displacement of the split position from the end to the beginning of the S4–S5 linker induces an increasing negative shift in activation voltage dependence, a reduced zg value and a more negative ΔG0 for current activation, an almost complete abolition of the activation time course sigmoid shape and a slowing of the voltage-dependent deactivation. Channels disconnected at the S4–S5 linker near the S4 helix show a destabilization of the closed state(s). Furthermore, the isochronal ion current mode shift magnitude is clearly reduced in the different splits. Interestingly, the progressive modifications of voltage dependence activation gating by changing the split position are accompanied by a shift in the voltage-dependent availability to a methanethiosulfonate reagent of a Cys introduced at the upper S4 helix. Our data demonstrate for the first time that alterations in the covalent connection between the voltage sensor and the pore domains impact on the structural reorganizations of the voltage sensor domain. Also, they support the hypothesis that the S4–S5 linker integrates signals coming from other cytoplasmic domains that constitute either an important component or a crucial regulator of the gating machinery in Kv11.1 and other KCNH channels.

Highlights

Voltage-gated potassium (Kv) channels play a crucial role for regulation of excitability in numerous cell types [3, 24, 63]
Since the Kv11.1 Y545 split channel showed most of the voltage-dependent properties of the continuous protein [33], these results challenged the traditional view of the S4–S5 loop acting as a mechanical lever to transduce the voltage-dependent motions of the voltage sensing domain (VSD) to the cytoplasmic gate at the bottom of S6
To obtain some additional insights about the mechanism involved in the coupling of the voltage sensor to the pore module in the presence of an interrupted S4–S5 linker, we generated a family of split channels in which the covalent link between the VSD and the pore domain (PD) was sectioned at different residues along the S4–S5 loop

Summary

Introduction

Voltage-gated potassium (Kv) channels play a crucial role for regulation of excitability in numerous cell types [3, 24, 63]. Our data suggest that by actively pushing the C-terminal end of the S4 helix and/or the initial section of the S4–S5 linker against the S5–S6 module, the closing of an otherwise intrinsically stable open pore can be favoured at negative transmembrane potentials. This process is influenced by other cytoplasmic structures such as the highly flexible N-tail of the VSD, that allosterically could affect different phases of a non-mechanical, but allosteric mechanism of gating [33]

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