Abstract
Voltage-gated channels open paths for ion permeation upon changes in membrane potential, but how voltage changes are coupled to gating is not entirely understood. Two modules can be recognized in voltage-gated potassium channels, one responsible for voltage sensing (transmembrane segments S1 to S4), the other for permeation (S5 and S6). It is generally assumed that the conversion of a conformational change in the voltage sensor into channel gating occurs through the intracellular S4–S5 linker that provides physical continuity between the two regions. Using the pathophysiologically relevant KCNH family, we show that truncated proteins interrupted at, or lacking the S4–S5 linker produce voltage-gated channels in a heterologous model that recapitulate both the voltage-sensing and permeation properties of the complete protein. These observations indicate that voltage sensing by the S4 segment is transduced to the channel gate in the absence of physical continuity between the modules.
Highlights
Introduction in the splitC-terminal permeation module of aS620T pore domain point mutation, known inactivation[27], reduced or to antagonize KV11.1 eventually abolished inactivation of the split channel (Fig. 8c), without affecting the voltage dependence of activation (Fig. 8c) or deactivation (Supplementary Fig. 2)
We demonstrate that at least for KCNH channels, the physical continuity between the voltage sensing and the pore modules is not necessary for voltage-dependent gating, challenging the classical view of a S4–S5 linker acting as a rigid mechanical coupler between them, and opening new questions about the nature of the molecular and functional interactions between the voltage-sensing and pore modules of the protein
To study the requirement of the integrity of the S4–S5 linker, we introduced a stop codon in KV10.1 after each residue in the linker (341–349: sequence LDHYIEYGA) and removed the rest of the channel sequence
Summary
Introduction in the splitC-terminal permeation module of aS620T pore domain point mutation, known inactivation[27], reduced (at 2 mM K þ ) or to antagonize KV11.1 eventually abolished (in 50 mM) inactivation of the split channel (Fig. 8c), without affecting the voltage dependence of activation (Fig. 8c) or deactivation (Supplementary Fig. 2). 41), which shifts the activation voltage dependence towards more negative values and accelerates activation of HERG channels, induced similar effects when performed in the split N-terminal half of the channel (Fig. 9 and refs 35,41). The voltage sensor of the initial half is able to confer near normal voltage-dependent properties to the assembled construct, and the functional properties of the pore domain leading to the characteristic voltage-dependent inactivation of HERG are maintained in the co-assembled channels. Our data unequivocally demonstrate that the permeation properties of the split channels are determined by the C-terminal domain, but it is the initial voltage sensor-containing module what crucially determines the voltage-dependent properties of the assembled protein. No active channels were recorded when the voltage sensor-containing N-terminal half of KV11.1 was co-expressed with the C-terminal half of KV10.1. Co-injection in the oocytes of the N-terminal voltagesensing module of KV10.1 (corresponding to residues 1–347, this last one in the middle of the S4–S5 linker) with the C-terminal
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